1.0 INTRODUCTION 1.1 Prevalence of fungal infection in ...studentsrepo.um.edu.my/3675/3/THESIS.pdf · 1.1 Prevalence of fungal infection in oral cavity Candida sp. inhabits various

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1.0 INTRODUCTION

1.1 Prevalence of fungal infection in oral cavity

Candida sp. inhabits various parts of the human system including the epidermis,

vagina, gastro-intestinal tract, nails and oral cavity (Williams et al., 2011). The disease

caused by Candida sp. has become a common disease in the late 19th and 20

th century

and its prevalence is still increasing worldwide as a result of multiple factors which can

facilitate the conversion of its commensal level to the parasitic level (Samaranayake et

al., 2009). According to Scardina et al. (2007), the risk factors that enhance the severity

of a candidal infection can be found widely in patient with impaired salivary gland,

drug abusers, high carbohydrate diet, smoking habits and Cushing‟s syndrome.

Candidal infection can occur in almost all human organs. However, it is the systemic

infection that can be much more severe and may lead to mortality. According to Leroy

et al. (2009), the mortality rate due to systemic infection of Candida sp. was up to 60%

and still increasing. The treatment of candidal infection can be difficult and most of the

diagnosis can only be achieved by autopsy. With the current incidence in Europe, there

has been increasing reports in candidemia of 5-folds within 10 years (Bassetti et al.,

2009). In a most recent study, candidal infection was also associated in oral cancer,

burning mouth syndrome, endodontic disease and taste disorder (Williams et al., 2011).

Candida albicans is the main causative agent of oropharyngeal candidiasis.

Researchers have however found that non-albicans species also contribute significantly

to the development of oral candidosis (Magaldi et al. 2001). Cases due to non-albicans

species are increasing in number and this has raised great concern to the society.

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In Malaysia, based on a survey in 1999, from a total of 1114 yeasts isolates from

the University of Malaya Medical Center (UMMC), 1.2% was identified as Candida

krusei (Ng et al., 1999). A case-control study among 100 healthy subjects in the

UMMC by Rasool et al. (2005) had shown a predilection for the Chinese infected by

Candida sp. while the Indian group was found to be the least infected. This is an

indication that the dispersion of Candida sp. may vary between ethnic groups. This is a

strong indication that the habit and background of each different ethnicity might have

influence on the dispersion. Candidal infection is still of a major concern although a

survey carried out in the Hospital of Kuala Lumpur had shown a decreased in candidal

isolation in the last four years (HKL, 2011). Despite this claim however, total isolates

of Candida albicans was found to increase every year.

1.2 Candida krusei in oral infection

Candida krusei has been identified as the fifth medically dominant pathogen

after Candida albicans, Candida glabrata, Candia kefyr and Candida parapsilosis

(Samaranayake and Samaranayake, 1994). Together with Candida glabrata, Candida

krusei were recognized as a common pathogen isolated from the blood stream

worldwide (up to 25%) (Quindós et al., 2008). Candida krusei was also classified as an

important pathogen involved in the escalating serious infections involving

immunocompromised patients (Samaranayake, 1997; Singh et al., 2002; Hakki et al.,

2006; Pfaller et al., 2008; Quindós et al., 2008). Candida krusei was associated with

the increased of nosocomial infection within the last two decades and has remained the

causative agent of morbidity and mortality in immunocompromised patients (Muñoz et

al., 2005).

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In the oral cavity, Candida krusei is known as one of the pathogenic yeasts

associated with oral candidosis. It persists in the form of patchy to confluent, whitish

pseudomembranous lesion. The site of infection is often composed of epithelial cells,

yeasts and pseudohyphae. Oral candidosis associated with Candida krusei has been

reported to increase widely in adolescent and children infected with oral cancer.

Compared to Candida glabrata and Candida tropicalis the occurrence of Candida

krusei in oral cancer patients was found to be higher (Gravina et al., 2007).

Candida krusei is an opportunist with multi-drug resistance and this include

towards fluconazole, a drug often prescribed for the treatment of candidal infection

(Samaranayake, 1997; Furlaneto-Maia et al., 2008). Candida krusei is however still

susceptible towards flucytosine voriconazole, echinocandins (caspofungin,

anidulafungin and micafungin) and amphotericin B at least until 2004. Later studies

then showed a reducing susceptibility incidence towards Candida krusei in oral

candidosis as a consequence of heterozygous mutation which had altered the sensitivity

of the yeast (Pfaller et al., 2004; Pfaller et al., 2008). In addition, treatment with

amphotericin B was found to be delayed in Candida krusei compared to Candida

albicans infections. A higher concentration of amphotericin B was also noted at ≥ 1

mg/kg of body weight per day in order to eliminate the development of Candida krusei.

Several reports have also discovered that there is decreasing susceptibility of Candida

krusei towards caspofungin, anidulafungin and micafungin (Hakki et al, 2006).

Data from a surveillance program in 2001 to 2005 in the Asia-Pacific region has

reported that, 1.3% of 137,487 isolates of Candida sp. from the Asia-Pacific region was

found to be Candida krusei (Pfaller et al., 2008). Malaysia had the highest number of

candidal isolates out of 8 other countries which include Australia, China, India,

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Indonesia, South Korea, Taiwan and Thailand. Malaysia was found to have as high as

39.54% candidal isolates and out of this candidal population, 1.3% isolates were

Candida krusei. A decreasing susceptibility of Candida krusei towards voriconazole

was also reported compared to Candida albicans. Based on a worldwide observation,

strains from the American Latin regions had showed the lowest susceptibility towards

voriconazole, which contradicts an analysis carried out in the South-East Asean

countries that about 75% of Candida krusei is still susceptible towards the anti fungal

agents (Pfaller et al., 2008).

1.3 Rationale of study

Candida krusei was classified in several studies as an emerging pathogen,

especially in immunocompromised patients (Merz et al., 1986; Samaranayake, 1997;

Abbas et al., 2000). In neutropenic patients, report has shown that an infection by

Candida krusei can lead to high mortality especially in leukemia patients who are

receiving fluconazole as prophylaxis (Abbas et al., 2000). Many therapies using

fluconazole and also caspofungin were failed in reducing the infection (Fleck et al.,

2007). This phenomenon has increased the awareness of researchers to focus on the

virulence factors of Candida krusei in the attempt to reduce the disease caused by the

pathogen. One of the most important virulent factors of Candida sp. is the ability to

switch its phenotypic in order to survive in an unfavourable growth environment

(Haynes, 2001). Thus, it is important to validate the ability of Candida krusei in

phenotypic switching and adherence. The understanding on this two characteristics

shed information on the pathogenicity of Candida krusei in causing candidal infection.

Candida sp. is also able to adhere firmly to hard surfaces of dentures. Candidal

adherence can occur either on the soft tissue such as the buccal and palatal surfaces or

on the hard tissue surface of dentures (Haynes, 2001). The ability to attach to hard

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surfaces is actually the key factor in initiating candidal infections. In addition, this

research is also targeted at assessing the antifungal activities of commercialized

antifungal agents and two plant extracts against Candida krusei. Nigella sativa and

Piper betle are two plants frequently used in traditional practices to cure various types

of illnesses. A positive antifungal property would enable these plants to be further

tested for use as antifungal agent.

1.4 Hypothesis of study

Phenotypic switching affects the biological properties of Candida krusei.

1.5 Aims of study

The aims of study are:

1) To determine the phenotypic switching ability of Candida krusei.

2) To determine the effect of phenotypic switching on the biological properties

of Candida krusei.

3) To evaluate the consequences of phenotypic switching on the susceptibility

towards commercialized antifungal agents and plant extract (Nigella sativa

and Piper betle).

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2.0 LITERATURE REVIEW

2.1 Oral Candida

2.1.1 Candida as a component of oral ecosystem

Candida sp. was identified as a common member of the oral microflora and was

estimated at 40% to 60% of the microbial population in the oral cavity. It can be

present either as transient or permanent colonizer in the oral cavity (Mitchell, 2007;

Thein et al., 2007). It is also recognised as an opportunistic microorganism that is able

of causing oral diseases such as oral candidosis (Marsh and Martin, 2009).

2.1.2 Colonization sites

Candida sp. is identified to colonise several host cell types including epithelial,

endothelial and phagocytic cells. In the oral cavity, Candida sp. prefers to colonise

several surfaces including the buccal and labial mucosa, dorsum or lateral borders of

tongue, hard and soft palate regions, tooth surfaces and denture-bearing areas (Cannon

et al., 1995; Siar et al., 2003). This colonising ability is contributed by factors

including the ability of the oral candidal species to produce specific enzymes such as

agglutinin-like proteins and integrin-like protein that lead to the formation of biofilm on

oral surfaces. In addition, other factors which influence the colonisation of Candida sp.

are the reduction of salivary flow, low salivary pH, trauma, carbohydrate-rich diets and

epithelial loss (Siar et al., 2003; Marsh, 2006).

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2.1.3 Growth requirement and susceptibility

2.1.3.1 Influence of oral fluids

Saliva provides moisture and helps in lubricating the oral cavity. It also

promotes the formation of thin film approximately 0.1 mm deep over all external

surfaces in the oral cavity. Saliva is produced by the major and minor salivary glands.

The major salivary glands consist of paired parotid, submandibular and sublingual

glands; whereas, the minor salivary glands are found in the lower lip, tongue, palate,

cheeks and pharynx. The chemical composition of secretions from each gland is

different; however, the major role of the whole saliva is to maintain the integrity of

teeth by clearing off food debris and buffering the potential damaging acids produced

by the oral biofilm or dental plaque. Bicarbonate, phosphates and peptides are

examples of buffering agent in the saliva which gives normal saliva a mean pH of 6.75

to 7.25 (Marsh and Martin, 2009).

The flow rate of saliva is under the influence of circadian rhythms where the

lowest flow often recorded during sleeping. Low flow rate of saliva reduces the

protective function of saliva and increases the colonisation and development of

microorganisms including Candida sp. Salivary composition is also affected by

circadian rhythms for example the total concentration of protein in whole saliva during

resting time is estimated at 220 mg/100 mL, whereas the total protein in stimulated

saliva is estimated at 280 mg/ 10 mL. This different amount of protein may affect the

distribution of the normal microflora in the oral cavity as some proteins are known to

serve as receptors in the colonisation of microorganisms to the saliva-coated surfaces of

the teeth (Marsh and Martin, 2009). Protein and glycoprotein such as mucin in the

saliva act as the primary source of nutrients for resident microflora including the

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candidal species. In addition to adherence, some proteins are also involved in host‟s

defence mechanism by aggregating exogenous microorganisms, hence, facilitating their

clearance from the mouth during swallowing or spitting.

In addition to saliva, the gingival crevicular fluid (GCF) in the oral cavity can

also influence the colonisation of oral Candida sp. The flow of GCF is slow at healthy

sites but increased drastically at areas with gingivitis by 147% and up to 30-fold at areas

with advanced periodontal diseases. GCF also has a role in the development of

subgingival plaque around and below the gingival margin. Among the host defence

components in GCF are includes IgG and neutrophils. GCF also contains higher total

protein compared to saliva. Thus, GCF is capable of providing nutrient sources to

several commensal microorganisms in the oral cavity (Marsh and Martin, 2009).

2.1.3.2 Influence of nutrients

Candida sp. is a chemoheterotrophic organism that requires carbon and nitrogen

for growth. According to Madigan and Martinko (2006), the mutual interaction of

carbon and nitrogen is important in the metabolism of microorganisms. Carbohydrates

are the most readily utilised form of carbon in both oxidative and non-oxidative way.

Thus, the presence of carbohydrates influenced the colonisation of Candida sp. in the

oral cavity. Certain carbohydrates such as sucrose and glucose have been shown to

increase the adhesion potential of Candida albicans towards hard and soft surfaces of

oral cavity. Glucose is an acid promoters which will lead to the reduction of pH in oral

environment with consequence of activation of acid proteinases and phospholipases

which then enhances the adherence of Candida sp. In addition, the production of

mannoprotein surface layer in the glucose presenting environment has been shown to

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assist the adherence of Candida sp. including Candida krusei in the oral cavity (Marsh

and Martin, 2009).

Candida sp. has nitrogen content of around 10% of their dry weight (Wai,

2009). The source of nitrogen is usually provided by organic compounds which can be

easily found in the oral environment. Nitrogen is also determined as the main

stimulatory factor in yeast extract as it encourages bio-stimulation on microbial growth.

2.1.3.3 Influence of body temperature

The optimum growth temperature for candidal species including Candida krusei

has been shown to range between 30 ºC to 37 ºC (Singh et al., 2002). This range of

temperature is considered as the optimum temperature of various pathogenic

microorganisms in the oral cavity. Any alteration in the normal body temperature may

however influence the competitiveness among the normal microflora which will then

enhances the development of opportunistic microorganisms such as Candida sp.

Nonetheless, many experimental assays were conducted at 37 ºC and this is generally

accepted as the standard incubation temperature for candidal species (Marsh and

Martin, 2009).

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2.1.4 Pathogenic determinants of Candida

The virulent factors of each different candidal species are not similar and can be

a comparative factor between each different species (Haynes, 2001). Among the

important virulent factors of Candida sp. include phenotypic switching, adherence to

host cells, cell surface hydrophobicity and enzymes production.

2.1.4.1 Phenotypic switching

Phenotypic switching is an important technique of survivorship of Candida sp.

within a growth environment including the oral cavity. Switching ability promotes

Candida sp. to adapt in suppressed environment and develop as the dominant host

microniches. Candida sp. can undergo reversibly high frequency of phenotypic

switching which increases and ensures the survivability of the pathogen (Haynes, 2001).

The details of this virulence factor will be further discussed in section 2.3.

2.1.4.2 Adherence ability

The adherence ability of Candida krusei is an important factor in the initiation

of oral candidosis. Adherence can occur either on the hard tissue surfaces such as the

teeth and palatal surface or smooth surfaces such as the buccal and lingual surfaces

(Samaranayake et al., 1994). Several characteristics of candidal species which

contribute to the adherence on these surfaces include the formation of pseudohyphae

and extracellular matrix.

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A single filament hypha (plural, hyphae) is a long branching filamentous

structure of fungus which can be found easily in the developmental phase of Candida

sp. (Madigan and Martinko, 2006). It is classified as the main mode of vegetative

fungal growth and consists of one or more cells which are surrounded by tubular cell

walls made of chitin. Hyphae usually grow together to form a compact tufts which are

known as mycelium. Hyphae formation is usually referred to the germination of fungi.

However, it is also involves in the colonisation of the target host. Pseudohyphae are

distinguished from the true hyphae by their method of growth which lacks cytoplasmic

connection between the cells. The pseudohyphae of Candida sp. are usually found to

possess incomplete budding blastoconidia whereby cells remain attached to the mother

cells after division. Candida albicans and Candida krusei has been recognised to

develop pseudohyphae which adhere to the monolayer of human epithelial cells (Soll,

1992; Dede and Okungbowa, 2009).

In many cases, extracellular matrix is also produced by oral microorganisms.

Extracellular matrix is a network of non-living mass which provides support to cells

including the Candida sp. The presence of extracellular matrix provides support to cells

attachment. This anchorage property assists the colonisation of candidal species to hard

tissue surfaces and thus, contributes to the formation of biofilm. When in a biofilm, the

resistance of candidal species towards various antifungal agents including amphotericin

B might be increased (Hawser et al., 1998).

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2.1.4.2.1 Dental biofilms

Dental biofilm is defined as a thin layer comprising of various communities of

microorganisms including bacteria, fungus and yeast that are attached on tooth surfaces

and on the surface of prosthesis including dental acrylic surfaces and human epithelial

cells (Holmes et al., 2002; McCarron et al., 2004). Microorganisms in the biofilm are

enclosed in a matrix of extracellular polymeric substance (EPS) (Branchini et al., 1994;

Samaranayake et al., 2002). This biofilm provides protection to the microorganisms

and facilitates the interaction among themselves with the contribution of biochemical

substances such as catalase and superoxidase dismutase (Socransky and Haffajee, 2002;

Marsh, 2004; Marsh, 2006). The development of biofilm is dependent on the dietary,

salivary and oral environmental factors that interact with the microorganisms within the

community of biofilm.

The formation of biofilm has been shown to reduce the susceptibility of

microorganism to antimicrobial agents which may then lead to the increase in

pathogenicity (Marsh, 2006). This phenomenon is suggested to occur due to the

restriction of the antimicrobial agents to penetrate the matrix of the biofilm which then

reduces the susceptibility of the target microorganism (Gilbert et al., 2002). In some

cases, the resistance of a pathogen in a biofilm can increase to 1000-fold towards an

antibiotic (Stewart and Corteston, 2001).

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2.1.4.2.2 Development of dental biofilms

The development of dental biofilm involves several stages which are the

acquired pellicle formation on the teeth surface; adhesion, reversible and irreversible

interactions between the pellicle and the colonising microbes; co-aggregation between

microorganisms; and detachment of microbes from the oral surfaces. These sequences

of events may eventually form a structural and functional organised microbial

community that if allowed to accumulate, may enhance the potential of periodontal

disease and dental caries (Marsh, 2004; Wan Nordini Hasnor, 2007).

The acquired pellicle formation is the formation of a thin, acellular layer which

works as the receptor of the attachment of the early plaque colonies such as

Streptococcus mitis, Streptococcus oralis and Streptococcus sanguinis. There are two

phases involved in the formation of the acquired pellicle which are the adsorption of

discrete protein of low molecular weight to the enamel surfaces followed by the

adsorption of protein aggregates of high molecular weight (Hannig, 1999).

The adhesion, both the reversible and irreversible adsorption properties of the

microorganism is a pioneer stage in the development of dental biofilm. Research has

shown that there is a reversible interaction which involves long-range physico-chemical

forces between the microbial and acquired pellicle on the oral surfaces (Marsh and

Martin, 1999). The net negative charge of the bacterial cell wall will interact with the

negative charged glycoprotein on the pellicle through a divalent cation bridge while, the

lipophilic adhesin of the microbial cell wall will recognise the hydrophobic receptors on

the ephitelial cell (Schonfeld, 1992). The van der Walls and electrostatic repulsion

forces which produce a weak area of net attraction facilitates reversible attraction

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between the microorganisms and the oral surface area. On the other hand, irreversible

interactions involve short range interactions with specific physico-chemical forces

between adhesins and the receptors on the surface area of the microbial cell surface and

the acquired pellicle, respectively. Streptococcus oralis and Streptococcus gordonii are

two examples of microorganisms involved in irreversible interaction that bind to

mucoglycoprotein of the acquired pellicle (Murray, 1992).

Subsequent to the colonisation of the early plaque bacteria to the acquired

pellicle, co-aggregation or co-adhesion of other microorganisms will takes place. This

is a process of microbial adhesion involving the late colonisers on the early colonizer of

dental biofilm. It is a phenomenon of cell-to-cell recognition of genetically distinct

partner cell types (Marsh and Martin, 1999). The co-aggregation can be facilitated

either through intrageneric such as the interaction between streptococci and among

Actimomyces (Streptococcus sanguis and Actinomyces sp.) or intergeneric such as the

interaction between Streptococci and Actinomyces (Streptococcus sp. or Actinomyces

and Prevotella sp.). Candida krusei has been found to be involved in co-aggreagation

with Streptococcus mutans, Streptococcus sanguis and Streptococcus salivarius in the

present of sucrose (Kiyora et al., 2000). Protein such as lectins is usually involves in

co-aggregation. This carbohydrate-binding protein will attach to the carbohydrate-

binding protein receptors of other cells which then contribute to the increased thickness

of the dental biofilm.

Once a climax community is achieved in the biofilm, detachment of some

microbes may occur in the final process oral biofilm development. The microorganism

is released from the matrix of biofilm to the fluid surrounding the biofilm a process

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which have been reported to be facilitated by several enzymes such as protease (Hunt et

al., 2004), fluid shear stress (Stoodley et al., 2001), multivalent cross-linking cation

(Caccavo et al., 1996) and microbial growth status (Jackson et al., 2002). This process

of detachment will however help the microorganism to colonise other surfaces in the

oral cavity. An example of microorganism involved in the detachment process from the

oral biofilm is Prevotella loescheii which produces protease that hydrolyse its fimbrae-

associated adhesion which is important in its co-aggregation with Streptococcus oralis

(Cavedon and London, 1993; Marsh and Martin, 1999).

2.1.4.3 Cell surface hydrophobicity

The virulence factor of Candida krusei can also be observed from the cell

surface hydrophobicity characteristic. This factor is classified as one of the most

important adherence mechanisms in the colonisation of the host surface. Candida

krusei is more hydrophobic compared to other medically important Candida sp.

(Samaranayake et al., 1993). Candida krusei was reported to possess the same

hydrophobicity level with Candida glabrata and Candida tropicalis but is more

hydrophobic compared to Candida albicans and Candida parapsilosis.

2.1.4.4 Enzyme

Hydrolytic enzymes of Candida sp. have been reported to contribute to the

pathogenicity in causing oral diseases such as oral candidosis. The enzymes include

aspartyl proteinase, phospholipases, lipases, phosphomonoesterase and hexosaminidase

(Williams et al., 2011). Among these enzymes, aspartyl proteinase has attracted most

interest and is widely considered to be central in the development of candidal infection.

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Aspartyl proteinase is a hydrolytic enzyme which is secreted by the transcription and

translation of sphingolipid activator protein (SAP) gene. This enzyme has the ability to

attack host and also contributes as a defence system of yeast. Examples of candidal

species possessing this enzyme are Candida albicans and Candida krusei

(Samaranayake, 1994).

Another important hydrolytic enzyme is phospholipase which is identified as an

enzyme that attacks the host tissue. This enzyme activity has been observed in many

fungal pathogens including Candida sp. There are 4 types of phospholipases which are

type A, B, C and D. Phospholipase A and C can be found in Candida albicans;

however, there is no evidence that shows the activity of phospholipase B and D in

candidal species (Samaranayake, 1994). Phospholipase A can attack cell membranes

and can be easily found on the cell surface especially at the sites of bud formation.

Hence, the enzyme activity can be enhanced when the hyphae are in direct contact with

the host tissue (Williams et al., 2011).

2.2 Candida krusei

Candida krusei is classified as a facultative saprophytic fungus that is

infrequently isolated from the human mucosal surface area (Do Carmo-Sousa, 1969;

Odds, 1988; Thein et al., 2007). This pathogenic yeast has been detected as an oral

commensal and represented between 10% to 15% of yeasts isolated from the oral cavity

of human. Since the 1960s, Candida krusei has emerged as a pathogen associated with

the development of oral candidosis (Samaranayake, 1994).

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2.2.1 Taxonomic status

Candida sp. is classified under the family of Cryptococcaceae as imperfect

fungi. The family of Cryptococcaceae includes the genera Torupsilosis and

Cryptococcus. The strain is recognised as the causative agent of thrush which infects

the mucosal layer including tongue, lips, gums or palate. The association of this

numerous generic and variable species of microorganisms and the lesion formed in the

oral cavity is called “thrush fungus” (Odds, 1988).

Candida krusei has been discovered since 1839 by Langenbeck and was firstly

isolated from the buccal epithelial layer in a typhus patient. However, it was

unclassified as pathogenic to human until 75 years later when Castellani found that the

strain was actually a commensal in warm-blooded animals (Castellani, 1912;

Samaranayake and Samaranayake, 1994). In general, the yeast morphology of Candida

krusei comprises of asexual and sexual species. The sexual form was renamed as

Issatchenkia orientalis whereas the asexual form had remained as Candida krusei (Odds

and Merson-Davies, 1989).

2.2.2 Biology of Candida krusei

Candida krusei is classified as yeast that possesses elongated cell shape with

“long grain rice” appearance. The appearance of this cell is shared with Candida kefyr

or also known as Candida pseudotropicalis (Samaranayake, 1997). The measurement

of Candida krusei is approximately 2.2 to 5.6 x 4.3 to 15.2 µm. It forms spreading

colony with matt or rough whitish yellow surface on SDA which in a way enable us to

identify it directly from morphological observation (Samaranayake and Mac Farlane,

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1990). The ultra structure of Candida krusei comprised of a six-layered cell wall with a

few intra-cytoplasmic organelles including small vesicles, lipid droplets, ribosomes and

glycogen-like particles (Joshi et al., 1975). The multilayered cell wall of Candida

krusei comprise of an outer irregular coat of flocculent material, an electron dense zone,

a granular layer, less granular layer, a thin layer of dense granules and another sparsely

granular layer outside the trilaminar cell membrane (Samaranayake and Samaranayake,

1994).

Candida sp. may exist in three morphological forms which are blastospores

(yeast-like ovoid cells), filamentous hyphae and chlamydiospores (dormant phase of the

microorganism). Chlamydiospores is a thick-walled spherical cell with approximately

10 µm in diameter (Melville and Russells, 1975). In many causes however, Candida

krusei is usually found in only two basic morphological forms which are the yeast and

pseudohyphae forms. Pseudohyphae are important in the adherence of cells to the

surfaces of the host (Soll, 1992; Dede and Okungbowa, 2009). Both characteristics may

however appear simultaneously thus making it difficult to differentiate between these

two basic characteristics.

Candida krusei can grow in an environment with temperature ranging from 35

ºC to 45 ºC (Odds, 1988). A characteristic that gives advantage to Candida krusei is

that it can grow in vitamin-free media, which is a major contrasting feature from the

other clinically important Candida sp. It is also reported that from a wide panel of

carbohydrates component Candida krusei can only ferment glucose (Barnett et al., 1983

and Silverman et al., 1990) which is also displayed by Candida pintolopesii. It has

been shown that when saliva is supplemented with glucose, a number of short-chain

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carboxylic acids such as acetate, pyruvate, succinate, propionate, lactate and formate

will be formed. Another feature that adds to the advantage of Candida krusei is that it

can produce acetoin which can be utilised when the growth environment is exhausted of

carbon sources (Lategan et al., 1981).

2.3 Phenotypic switching ability of oral Candida

Two mechanisms were postulated to be involved in the ability of Candida krusei

to survive and adapt in a suppressed environment. First is by undergoing mitotic

recombination and second is by carrying out phenotypic switching. A direct

consequence of mitotic recombination is the lost of heterozygosity throughout the entire

genome. This deletion of genome however affects the viability of Candida sp.

especially in the multiple changing conditions (Vargas et al., 2004). Phenotypic

switching, on the other hand is a phenomenon that occurs as a result of changes in the

growth environment. A severely suppressed growth condition may lead to high

frequency switching in candidal cells. This adaptation is associated with the alteration

of gene expression which eventually may lead to alteration of adhesiveness,

susceptibility and the resistance of candidal cells to phagocytosis and

polymorphonucleur leukocyte. This mechanism of action does not involves deletion of

any candidal genome thus, the heterozygosity of the entire genomic are well maintained

(Martin and Marsh, 2009).

Phenotypic switching is identified as one of the important virulent factors in

Candida albicans (Anderson et al., 1987; Soll, 1992; Jones, et al., 1994; Vargas, et al.,

2004) and Candida glabrata (Lackhe et al., 2000; Lackhe et al., 2002). The

significance of the switching strategy is in a way similar to the human immunity

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function whereby it is aimed to counter threats in the host‟s environment. Until now,

there is no report that highlights the ability of Candida krusei to undergo phenotypic

switching. However, scientist has suggested that phenotypic switching mechanism

enhances the survivability of Candida sp. by rapidly changing its phenotype as an

adaptive response to the suppressed environment (Hellstein et al., 1993).

Phenotypic switching may influence the normal physiological growth of

candidal species such as Candida albicans (Vargas et al, 2004). Under the smooth

white and wrinkled phenotypes, Candida albicans has been shown to exhibit faster

growing colonies than when it exhibited a heavy myceliated and ring phenotype. In

addition, phenotypic switching is also discovered to be able to alter the adhesiveness

properties of Candida sp. (Kennedy et al., 1988). Furthermore, this virulence attribute

may also induce the formation of tube and pseudohyphae in Candida sp. which then

enhance the adherence capacity of the candidal strains (Lackhe et al., 2002).

2.4 Management of candidal infection

2.4.1 Antimicrobial agents

Antimicrobial agents are described as drugs which selectively help to eliminate

microbial pathogens from host cell due to toxicity mechanism. There are three

categories of antifungal agents available for the treatment of candidosis. They are

polyenes, azoles and the DNA analogue 5-fluorocytosine. Examples of polyenes are

nystatin and amphotericin B, while the azoles include miconazole, fluconazole,

clotriminazole, ketoconazole and itraconazole. The principal of antifungal agents used

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against yeast infections in the oral cavity belongs to the first two categories which are

the polyenes and the azoles (Samaranayake & Ferguson, 1994).

2.4.1.1 Disinfectant

2.4.1.1.1 Chlorhexidine

Chlorhexidine (CHX) has antifungal and antibacterial properties. The principle

of treatment with CHX is based on the rapid absorption of the chemical component into

the surface of the microorganisms which increases the permeability of the cell

membrane. As a result, it causes precipitation of the cytoplasmic content which then

kills the microorganism directly (Davies, 1973). It is also widely used in the treatment

of oral candidosis (Budtz-Jorgansen and Loe, 1972; Kulak et al., 1994). Regular

rinsing with chlorhexidine helps in the treatment of this disease (Langslet et al. 1974).

However, the prolonged usage of CHX may stain the dental hard tissue surfaces

brownish.

2.4.1.2 Chemical-Based agents

2.4.1.2.1 Azoles

These antifungal agents have five-member of organic rings containing two or

three nitrogen molecules (Anil, 2002). Azoles are categorised as N-1 substituted

imidazoles and triazoles. Imidazoles that are usually used clinically includes

ketoconazole and miconazole while the triazoles include the itraconazole and

fluconazole. Triazoles such as fluconazole are inhibitor of cytochrome p-450 enzyme

which is involved in the general synthesis of fungal cell membrane. The principal

22

action of azoles is through the conversion of 14-α-methylsterol to ergosterol within the

fungal membrane as such conversion may lead to the blockage of 14-α-demethylation

step in the synthesis of ergosterol. As a result, the composition of ergosterol will

deplete whereas the 14-α-methysterol will accumulate and becomes permeable to the

intracellular constituents. This target process is known as 14-α-demethylase.

Imidazoles on the other hand may interfere with the fungal oxidative enzymes which

then lead to lethal accumulation of hydrogen peroxide in the cell (Anil, 2002).

2.4.1.2.1a Fluconazole

Fluconazole is an antifungal agent which is commonly used in the treatment of

candidal infection such as oral candidosis. It is a water-soluble chemical compound

which can be found in the form of tablet, oral solution and saline-based intravenous

solutions (Anil, 2002). In a bulk powder form, it appears as a white crystalline powder

with slight solubility water and alcohol. The absorption of fluconazole has been

reported to be unaffected by food or gastric acidity (Lim et al., 1991; Debruyne and

Ryckelynck, 1993; Zimmermann et al., 1994).

Fluconazole has been useful in the prevention of Candida albicans-associated

endocarditis and diseases caused by Candida parapsilosis and Candida tropicalis.

However, this antifungal agent was found to be ineffective towards Candida krusei and

Candida glabrata (Van‟t Wout, 1996; Samaranayake, 1997; Venkateswarlu et al., 1997;

Singh et al., 2002). In other species, fluconazole exerts fungistatic affects. This

fungistatic azoles is often used for lifetime therapy of AIDS patients. This is of concern

as long-term usage may lead to drug resistance especially when switched candidal

species are involved (Baily et al., 1994).

23

2.4.1.2.1b Voriconazole

Voriconazole is a triazole derivative of azoles with a wide range of effectiveness

in the treatment of fungi including Candida sp., Aspergillus sp. and Cryptococcus

neoformans. In comparison to fluconazole therapy, voriconazole exhibited 1.6 to 160-

fold greater inhibition of ergosterol P-450-dependent α-demethylase in Candida

albicans, Candida krusei and Aspergillus fumigates (Sheehan et al., 1999).

Voriconazole is more potent in inhibiting the growth of Candida krusei compared to

fluconazole with 16-fold higher than the treatment on Candida albicans (Fukuoka et al.,

2003).

2.4.1.2.2 Polyenes

The polyenes are antifungal drugs that target the cell membrane containing

ergosterol. Polyenes include nystatin and amphotericin B which are categorised as the

amphipathic; having both hydrophobic and hydrophilic sides. It acts as an agent that is

able to intercalate into membrane layer and forming channels causing potassium ions to

leak out of the cell and destroy the proton gradient of Candida sp. (Vanden Bossche et

al., 1994).

2.4.1.2.2a Amphotericin B

Amphotericin B is a polyene which posses both fungicidal and fungistatic

activities that are broad-spectrum against Blastomyces dermatidis, Coccidioides imnitis,

Crytococcus neuformans, Histoplasma capsulatum, Paracoccidioides brasiliensis,

Sporotrichium sp., Candida glabrata and Candida albicans except Candida lusitaniae

which is resistant. This topical antifungal agent is usually used in treating primary oral

24

candidosis and used as an adjunct to parenteral therapy in secondary candidosis with

manifestations of both systemic as well as on oral mucosal surfaces (Samaranayake et

al., 2009).

Amphotericin B acts on the sterol on the cell walls of the target cells which then

damages the cell walls and releases the ionic content including potassium and glucose.

This phenomenon leads to the inhibition of glycolysis which then inhibits the growth of

the candidal species (Anil, 2002).

2.4.1.2.2b Nystatin

Nystatin was discovered in 1949, obtained from Streptomyces noursei that was

found in the soil sample of a farm in Virginia. It is found to be the most established

antifungal agent that is effective in the treatment of superficial fungal infection caused

by Candida sp. Nystatin can damage the cell membranes of yeasts by altering the

permeability (Kerridge, 1986). The reaction starts when nystatin binds to the sterol

component in the membrane of the yeast and alters the permeability. Nystatin has the

ability to treat superficial fungal infection caused by Candida sp. It has been shown to

exhibit both the fungistatic and fungicidal activities. However, nystatin is a poor

therapy for oral candidal infection. It is poorly absorbed by the host and usually it is

passed unchanged through the gastrointestinal tract (Anil, 2002).

Furthermore, it is discovered that nystatin can suppress the adhesion of Candida

albicans to cells of the buccal epithelium (Macura, 1987; Vuddhakul et al., 1988; Abu-

el Teen et al., 1989). Nystatin can also inhibit the formation of germ tube which is

25

known as the virulence factor of selected candidal strains such as Candida albicans and

Candida dubliniensis.

2.4.1.3 Plant-based agents

2.4.1.3.1 Family Piperaceae

Piper betle is a plant belonging to the Piperaceae which originated from the

South East Asia including India, Sri Lanka and Bangladesh. The betel leaf itself is

known as Sireh (Malay), Paan (Urdu and Hindi), Vetrilai (Tamil) and Ikmo

(Philippines). Piper betle plant is an evergreen plant with glossy heart-shaped leaves

with white catkin. Usually, the leave is chewed together with betle nut, lime and

gambier leaves. The nut gives the reddish colour to the saliva and thus darkens the

teeth. Betle leave is believed to be a folk medicine in the treatment of various diseases

including bad breath, headache, boils, conjunctivitis, itches, mastitis, mastoiditis and

ringworm (Chopra et al., 1956). The essential oil of Piper betle was reported to contain

antibacterial, antiprotozoan and antifungal properties. Research has shown that the

plant may produce bacteriostatic and fungistatic effects against Salmonella thyphi,

Escherichia coli and Candida albicans respectively (Indu and Ng, 2002; Guha, 2006).

Piper betle was found to be effective as anti-dermatophyte against Candida

albicans, Microsporum gypseeum and Trichosporon beigelii and phyto-pathogen such

as Sclerotium rolfsii, Alternaria solani and Phytophthora infestons (Rahman et al.,

2005). The extract of this plant was also identified as an important antioxidant which

scavenged the free radicals and detoxify the organism which then prevented

cardiovascular disease, cancers (Gerber et al., 2002; Serafini et al., 2002), Parkinson‟s

26

and Alzheimer‟s diseases (Di Matteo and Esposito, 2003). The leaf extract was

identified to inhibit radiation-induced lipid peroxidation. In addition, the extract also

increased the activity of superoxide dismutase which indicated the elevation of

antioxidant status in Swiss albino mice (Chourhury & Kale, 2002). Research has also

proved that the antioxidant component within Piper betle leaf was higher than tea

(Dasgupta and De, 2004). The active compounds which were identified from the

extract include chavicol, chavibetol, allylpyrochatichol, chavibetol acetate and

allylpyrochatichol diacetate. Various nutritional compounds has been identified to be

present in Piper betle extract which include vitamin A, vitamin B, iodine, iron, calcium,

potassium, tannin, riboflavin and carbohydrate. Furthermore, the leaf was also said to

contain enzymes such as diastase and catalase (Guha and Jain, 1997).

Piper betle crude aqueous extract has been reported to reduce the cell surface

hydrophobicity of Streptococcus sanguis, Streptococcus mitis and Actinomycetes sp.

(Fathilah et al., 2006). Hydrophobicity is an important mechanism which enhances the

adherence of pathogen to saliva-coated teeth surface.

27

Figure 2.1: The leaves of Piper betle.

Figure 2.2: Piper betle tree.

28

2.4.1.3.2 Family Ranunculaceae

Nigella sativa is a herbaceous plant which is known as fennel flower plants

derived from Ranunculaceae or Buttercup family. The maximum height of this plant is

about 60 cm with blue flower producing small-caraway black seeds (Khan, 1999; Al-

Jabre et al., 2003). The plant is also known as black cumin (English), shonaiz (Persian),

krishnajirika (Sanskrit), kalajira (Bangali), kalonji (Urdu and Hindi) and Habbatus-

sawda (Arabic). It is a common plant in the Middle East, Eastern Europe, Western and

Middle Asia. This plant has been identified as a remedy for many ailments since the

ancient times of the Egyptian, Romans and Greeks (Al-Jabre et al., 2003). The

medicinal parts of Nigella sativa were reported in the book of medicine Canon fi Tibb

by Avicenna which states that the black seed is a good medicine which acts as

expectorant, stimulates body‟s energy and helps in the recovery from fatigue and

dispiritedness. In the Quran, the black seed is known as the cure for any kind of known

disease except death (Al-Bukhari, verse 815). Besides medicine, it is also used as a

flavour for bread and pickles. Many active ingredients are found from Nigella sativa

which include thymoquinone, thymol, dithmoquinone, thymohydroquinone, carvacrol,

nigellicine, nigellidine, nigellimine-N-oxide and alpha-hedrin (Canonica et al., 1963;

Mahfouz and El-Dakhakhny, 1966; El-Alfy et al., 1975; Khan, 1999, Randhawa and

Al-Ghamdi, 2002; Al-Jabre et al., 2003).

Thymoquinone and thymohydroquinone present in the extract of Nigella sativa

were reported to have anti-inflammatory activities. Studies on rat perionatal mast cells

have shown that nigellone at low concentrations worked as an active inhibitor of

histamine which is produced during cell-antigen segregation. The action was created

due to the inhibition of protein kinase-C and the decrease of calcium concentration

29

which is involved in an inflammation mechanism. These results have suggested that the

nigellone can be used as an effective medicine in the prevention of asthma and allergic

condition (Zawahry, 1963; Chakravarty, 1993; Khan, 1999). Thymoquinone which is

the active component of Nigella sativa acted as anti-fungal agent towards Candida

albicans (Al-Jabre et al., 2003), Tricophyton rubrum (Al-Jabre et al., 2005) and

Aspergillus sp. (Al-Qurashi et al., 2007). Thymohydroquinone is also able to inhibit

the growth of Gram positive microorganism such as Escherichia coli. Diethyl-ether

extract of Nigella sativa was found to be effective on Staphylococcus aureus,

Pseudomonas aeruginosa and Escherichia coli. It was also discovered to act

synergistically with streptomycin and gentamycin (Atta-ur-Rehman et al., 1985; Morsi,

2000).

In the early 1960‟s, researchers found that the volatile oil of Nigella sativa

contained antimicrobial component (Toppozoda et al., 1965). The oil was found to

inhibit the growth of certain Gram positive bacteria such as Streptococcus aureus, Gram

negative bacteria such as Escherichia coli and fungi such as Candida albicans.

Furthermore, the fractionation process of the oil which produces the phenolic content

was found to increase the effectiveness of the oil up to ten thousand times and is non-

toxic to human (Toppozoda et al., 1965).

30

Figure 2.3: The seeds of Nigella sativa.

31

3.0 MATERIALS AND METHODS

3.1 RESEARCH MATERIALS

3.1.1 Chemicals

Decon 90 (Decon, England)

Dibasic sodium phosphate anhydrous powder (Sigma, USA)

Ethanol 95% (John Kollin Corporation, USA)

Germisep (Hovid, Malaysia)

Glutaraldehyde

Glycerol (Merck, Germany)

Monobasic potassium phosphate (Sigma, USA)

Osmium tetraoxide 1%

Phloxine B (Sigma, USA)

Potassium chloride (Sigma, USA)

Savlon (Johnson and Johnson, Malaysia)

Sodium chloride (BDH, England)

32

3.1.2 Glasswares

Beaker (Bibby, UK)

Conical flask (Pyrex, England)

Glass beads (3 mm diameter) (Merck, Germany)

Glass bottle (Schott, UK)

3.1.3 Consumables

Aluminium foil (Diamond, USA)

Blank disk (Oxoid, UK)

Bunsen burner gas (Campingaz, France)

Microtitre plate (96 wells) (Nunc, Denmark)

Petri dish (Brandon, USA)

Pipette tips (Appendorf, Canada)

33

3.1.4 Media

Bacto agar powder (BD, USA)

D (+) Glucose (Sigma, USA)

Mueller-Hinton (MH) agar powder (BD, USA)

Peptone powder (BD, USA)

Yeast extract powder (BD, USA)

3.1.5 Antifungal agents

Amphotericin B (250 µg/mL) solution (PAA, Germany)

Chlorhexidine digluconate (20%) (Sigma, USA)

Fluconazole (25 µg) discs (Oxoid, UK)

Nystatin (100 µg) discs (Oxoid, UK)

Voriconazole (1 µg) discs (Oxoid, UK)

3.1.6 Plant specimens

Nigella sativa (Durra, Syria)

Piper betle (Kedah, Malaysia)

34

3.1.7 Microbial test strain

Candida krusei (ATCC 14243), American Type Culture Collection, USA.

3.1.8 Microbial identification system

API 20 C AUX (BioMériux, France)

BIOLOG YT Micro Plates (BIOLOG, USA)

Mc Farland standards (BD, USA)

3.1.9 Equipments

Analytical balance, Denver XL-1810 (USA)

Analytical balance, Mettler AJ100J and Denver XL-1810 (USA)

Autoclave, HICLA VE HVE-50 (Hirayama, Japan)

Chiller, 4 ºC (Mutiara, Malaysia)

Compact digital camera (Olympus, Japan)

Digital Camera Light Reflection (DSLR) D90 (Nikon, Japan)

Electric drying cabinet, Weifo KD-112 (Weifo, Singapore)

Freezer, -80 ºC, Hetofrig CL410 (Hetofrig, Denmark)

Fume cupboard, Ductless Labcaire 4850 (Labcaire, England)

Haemacytometer (Marienfield, Germany)

35

Hotplate (Cimarec 3, USA)

Incubator (Memmert, Germany)

Laminar flow unit, ERLA CFM Series (Australia)

Light microscope (Nikon, Japan)

Micropipettors (Appendorf, Canada)

Microwave oven (Panasonic, UK)

Peristaltic pump (Bio-Rad Econo. Pump)

Scanning Electron Microscope (JOEL, Japan)

Spectrophotometer, Shimadzu UV160A (Shimadzu, Japan)

Speed vacuum concentrator, HETO/HS-1-110 (Denmark)

Sputter coater S150B (Edwards, USA)

Stereoscope (Olympus, Japan)

Thermocirculator E3500 (Polaron, UK)

Vortex mixer (Glas-Col, USA)

Water distiller (J Bibby Merit)

Water purifier system (ELGA, UK)

36

3.2 RESEARCH METHODS

3.2.1 Research outline

“

Figure 3.1: Schematic diagram of research methodology.

Candida krusei

Switched cells generations

1st

2nd 3

rd 4th

Unswitched cells generation

Antifungal

Response

Adherence to

Saliva Coated

Glass Surface

Susceptibility

Minimum

inhibitory

concentration

Growth curve

following

treatment

Minimum

fungicidal

concentration

Biological

Characteristics

Colony morphology

Biochemical

validation

Cell morphology

Ultrastructure

characteristic

Normal growth

curve

37

3.2.2 Preparation of broth media

3.2.2.1 Yeast extract potato dextrose (YEPD) broth

Table 3.1: Compounds required for making YEPD broth.

Materials g

D (+) Glucose 20

Peptone powder 20

Yeast extract powder 10

All the nutrients above were dissolved in 1 L of distilled water and boiled in a

microwave oven. The mixture was sterilized by autoclaving at 121 ºC for 15 minutes at

15 psi. The sterilized YEPD broth was kept in the 4 ºC refrigerator for later used within

a time period of a month.

3.2.2.2 YEPD broth supplemented with phloxine B

1 L of YEPD broth was prepared as above and 0.005 g of phloxine B (0.05%)

was added to the broth mixture. Phloxine B was mix thoroughly. The supplemented

broth was then sterilized by autoclaving at 121 ºC for 15 minutes at 15 psi and kept at 4

ºC for further use in the experiment. The prepared media was best to be used within a

month.

38

3.2.3 Preparation of agar media

3.2.3.1 Yeast extract potato dextrose (YEPD) agar

Table 3.2: Compounds required for making YEPD agar.

Materials g

D (+) Glucose 20

Peptone powder 20

Yeast extract powder 10

Bacto agar powder 20

All nutrients above were dissolved in 1 L of distilled water and boiled in a

microwave oven. The mixture was autoclaved at 121 ºC for 15 minutes at 15 psi. The

sterilized media was poured into sterile petri dishes and left to solidified.

Agar slants were also prepared by dispensing approximately 3 mL of the

sterilized agar into the sterile universal bottles and left to solidify on a slant bench

surface. The solidified YEPD agar plates and slants were stored 4 ºC refrigerator for

later use within a period of a month. The agar plates were stored in an inverse direction

until the next usage and were best used within a period of a month.

39

3.2.3.2 YEPD agar supplemented with phloxine B

1 L of YEPD agar suspension was prepared as above and 0.005 g of phloxine B

(0.05%) was added to the agar mixture and boiled in a microwave oven. Similar

sterilization procedure to section 3.2.2.1 was carried out. The sterilized media was then

poured into sterile petri dishes and left to solidified.

Agar slants were also prepared by dispensing approximately 3 mL of the

sterilized agar into the sterile universal bottles and left to solidify on a slant bench

surface. The solidified YEPD agar plates and slants were kept in the 4 ºC refrigerator

for later use within a month. The agar plates were kept in inverse direction until the

next usage.

3.2.3.3 Mueller-Hinton (MH) agar

38 g of MH agar powder were dissolved in 1 L of distilled water and boiled in a

microwave oven. The mixture was then sterilized following the procedure in section

3.2.2.1. The sterilized medium was then poured in sterile petri dishes and left to

solidified. All plates were kept at 4 ºC in an inverse direction and were best used within

a period of one month.

3.2.3.4 CHROMagar

47.7 g of CHROMagar powder were dissolved in 1 L of distilled water and

boiled in a microwave oven for 5 minutes. The mixture was then sterilized according to

the procedure in section 3.2.2.1 and poured in sterile petri dishes and left at room

40

temperature to solidify. All plates were kept at 4 ºC in an inverse direction. The plates

were best used within a period of one month.

3.2.4 Preparation of Candida krusei (ATCC 14243) stock culture

With reference to the manufacturers‟ instruction, an ampoule containing

lyophilised cells of Candida krusei (ATCC 14243) was added with 0.5 mL of sterile

distilled water to rehydrate the dry pellet. Following rehydration, 100 µL of the

suspension was then inoculated on to YEPD agar plate and incubated at 37 ºC for 24

hours (Lackhe et al., 2002).

3.2.4.1 Short term storage on agar slants

Several colonies of Candida krusei from YEPD agar plate were picked,

subcultured on YEPD slants and incubated overnight and stored at 37 ºC. It was then

stored at 4 ºC prior to use in the experiment.

3.2.4.2 Long term storage in 20% glycerol

Glycerol stock media is required to maintain cells‟ viability for long term

storage. Candida krusei strain from YEPD slant was inoculated into 5 mL of YEPD

broth and incubated overnight at 37 ºC. Following incubation, 800 µL of the growth

suspension was transferred into sterile microfuge tube which has been added with 200

µL of glycerol. The glycerol stock of Candida krusei was then stored at -80 ºC for long

storage purposes.

41

3.2.5 Preparation of Candida krusei switched cultures

Cells from the slant culture of unswitched Candida krusei was revived in YEPD

broth that has been supplemented with 5 mg mL-1

of phloxine B dye. This dye was

used to create a stress growth environment for the cells (Lackhe et al., 2002). The

suspension was then incubated for 4 to 5 hrs at 37 ºC. Following incubation, the

turbidity of the growth suspension was spectrophotometrically measured and

standardised to an optical absorbance 0.144 at a wavelength of 550 nm (106 cells mL

-1).

The suspension was then serially diluted to give a plate count of approximately 50 cells

each. The suspension was then spread evenly on the agar surface and incubated

overnight at 37 °C. The colony forming units (CFU/mL) were then examined.

Colonies exhibiting different characteristics from the normal were enumerated and

photographed. These colonies were considered as having a phenotypic switched from

the unswitched Candida krusei and designated as the 1st switched generation. Several

cells from the 1st switched generation were again subcultured on to another set of YEPD

(supplemented with phloxine B) agar plate and the whole procedure was repeated to

produce the 2nd

, 3rd

and 4th

generation of the switched cells. The CFU/mL of each

generation of unswitched and switched Candida krusei was calculated according to the

formula:

Total CFU/mL = Number of formed colonies

Dilution factors x volume used (mL)

The result was interpreted as the mean of CFU/mL and standard deviation (SD).

42

3.2.6 Determination of biological characteristics of Candida krusei

3.2.6.1 Colony morphology

3.2.6.1.1 YEPD agar

Candida krusei from the slant was inoculated into 0.5 mL of YEPD broth and

standardized to an OD of 0.144 at 550 nm. 100 µL of the suspension was then

inoculated on to YEPD agar plate and incubated at 37 ºC for 24 hours (Lackhe et al.,

2002). The morphology of Candida krusei which include the surface appearance,

margin, forms and elevation were observed and recorded.

3.2.6.1.2 CHROMagar

Using a sterile wire loop, Candida krusei was inoculated on CHROMagar using

single colony dilution streaking method. The plate was incubated at 37 ºC for 24 to 72

hours. Following incubation, the colour of the grown colonies were observed and

recorded.

3.2.6.2 Biochemical analysis

3.2.6.2.1 API 20 C AUX identification system

5 mL of distilled water was dispensed into each honey-combed wells of API 20

C AUX tray to provide a humid environment. The tray was labeled according to the

sample used. A suspension of Candida krusei was prepared and standardized at a

turbidity of 2 McFarland using sterile saline. 100 µL of the suspension was inoculated

onto YEPD agar and incubated at 37 ºC for 24 hours. Another 100 µL of Candida

krusei suspension was inoculated into C-medium which was supplied by the

43

manufacturer in the identification system kit. The suspension mixture was

homogenized gently to prevent the formation of bubbles. The homogenized suspension

was pipetted into each of 20 cupules on the test strip placed in the tray. The tray was

put in an incubator at 37 ºC for 48 to 72 hours. After incubation, the strip was

examined and the turbidity of each cupule was compared to the control sample. The

positive or negative outcomes of all cupules in the strip were compared to the table

provided by the manufacturer to confirm the species.

3.2.6.2.2 BIOLOG YT MicroPlates

The methodology was carried out according to the instruction provided by the

manufacturer. BIOLOG is an identification system which dependent on the substrate-

enzyme interactions of the microbial strain. An overnight cultured of Candida krusei

was suspended in sterile distilled water. Later, 100 µL of the cell suspension

standardized at 47% transmittance was inoculated into each well of the YT MicroPlates.

The YT MicroPlates were incubated at 26 ºC for 24, 48 and/or 72 hours. The metabolic

patterns were interpreted by Biolog‟s MicroLog 3 computer software which matched to

the library of species database.

3.2.6.3 Cell morphology

Candida krusei was inoculated in YEPD broth and incubated at 37 ºC for three

hours. Following this, one to two loopfuls of the suspension was placed on clean glass

slide. With circular movement of the loop, the suspension was spread evenly into a thin

area with approximately the size of 1 cm2. The smear was fixed by air-drying. The

smear was then gently flooded with crystal violet and left to stand for one minute, after

44

which the stained was washed with tap water. Later, the smear was flooded with iodine

and left for one minute and washed with tap water. 95% of ethyl alcohol was dropped

on the smear followed by immediate washing with tap water. A counterstain, safranin

was applied and left to stand for 45 seconds. Finally, the smear was then washed with

tap water and blot dried with tissue paper. The slide was then examined using a light

microscope at 100 x magnification under oil immersion.

3.2.6.4 Ultrastructural characteristic

A colony of C. krusei growing on YEPD agar was removed using a cork borer

and transferred into a sterile petri dish. The specimen was then fixed by immersing it in

glutaraldehyde and Sorensen‟s phosphate solution (1:1). After an hour, the specimen

was washed with Sorensen‟s phosphate and distilled water (1:1) and then post-fixed

with 1% osmium tetraoxide and distilled water (1:1) over a period of 14 hours at 4 ºC.

The specimen was then put aside for an additional one hour at 25 ºC under a laminar

flow. Following that, the 1% osmium tetraoxide was gently pipetted out and the

specimen was again washed with distilled water and put through a series of ascending

ethanol concentrations (10%-100%) to dehydrate the specimen. The specimen was then

immersed in 100% ethanol twice to ensure maximum elimination of water in the

samples. Gradual displacement of ethanol with acetone was then carried out (20

minutes each) using the following ratios of ethanol (EtOH) to acetone (v/v) with 3:1,

1:1 and 1:3.

Following that, the specimen was immersed in 100% acetone for four times, 20

minutes each time, followed by critical point drying (CPD) process using the Polaron

E3500. The specimen was then mounted on aluminium stubs and coated with gold

45

palladium using an Edward‟s sputter coater (S150B). The specimen was then examined

under the scanning electron microscope (SEM) (JEOL SEM) at 2000 x magnification.

Samples of the 1st, 2

nd, 3

rd and 4

th switched cells were also similarly prepared for SEM

examination.

3.2.6.5 Growth curves

In a sterilized Schott bottle, 50 mL of YEPD broth and 0.5 mL of Candida

krusei was added and the concentration of the suspension was standardized to 0.144

OD550nm (106 cells mL

-1). The suspension was then vortexed for 30 second and the

initial OD at 550 nm was recorded. The bottle was placed in a shaking water bath at 37

ºC and the growth of the cells was monitored by recording the OD reading at every one

hour interval. The OD reading was converted to CFU/mL and a graph of log10CFU/mL

versus incubation time was plotted. The experiment was stopped once the stationary

phase was achieved. The protocol was carried out in triplicate and repeated several

times to ensure reproducibility (Fathilah, 2004). The procedures were repeated and the

growth curves of the 1st, 2

nd, 3

rd and 4

th switched cell generations were also determined.

3.2.7 Determination of biological characteristics of switched Candida krusei

The determination of the biological characteristics of switched Candida krusei

was carried out following procedures in section 3.2.6.1 to 3.2.6.5. Observations with

regards to the colony and cell characteristics, the ultrastuctural changes and the growth

curves of the switched cells were recorded.

46

The recovery population of the 1st switched generation of Candida krusei was

determined from the percentage of the total CFU/mL of switched colony of the 1st

switched generation to the total CFU/mL of the unswitched Candida krusei. Whereby,

the recovery populations of the 2nd

to the 4th

switched generations of Candida krusei

were obtained from the percentage of the total CFU/mL of switched colony of Candida

krusei to the total CFU/mL of the previous switched generation of Candida krusei.

3.2.8 The effect of phenotypic switching on adherence to saliva-coated hard

surface

3.2.8.1 Preparation of phosphate-buffered saline (PBS) solution

Table 3.3: Chemical ingredients required for the preparation of Phosphate-Buffered

Saline (PBS).

Materials g

Sodium chloride 4

Potassium chloride 0.1

Dibasic sodium phosphate anhydrous powder 0.72

Monobasic potassium phosphate 0.12

The chemicals were dissolved in 1 L of distilled water and standardized at pH 7.

The solution was then autoclaved at 121 ºC for 15 minutes at 15 psi. The pH were

adjusted to pH 7.0 and then stored at 4 ºC.

47

3.2.8.2 Collection of stimulated whole saliva (SWS)

SWS was collected following method described by Holmes et al. (2002) with

some modification by Rahim et al. (2008). 25 mL of saliva was collected everyday

from a single volunteer with healthy oral condition in order to reduce any variation

between different individuals. Initially, the subject was required to rinse with distilled

water for 10 seconds to ensure the cleanliness of the oral cavity. The volunteer was

given a piece of rubber band to chew in order to stimulate salivary flow and SWS was

collected in a sterile ice-chilled test tube with the addition of 1,4-dithio-D,L-threitol

(DTT) to a concentration of 2.5 mM. The specimen was stirred slowly before

centrifugation at 17,000 g for 30 minutes. The supernatant obtained was filtered

through 0.2 µm pore low-protein binding filter (Supor® membrane) into a sterile test

tube. SWS was then stored at -20 ºC for use in the adherence study.

3.2.8.3 Adherence to saliva-coated hard surface

An artificial mouth model named the Nordini‟s Artificial Mouth (NAM) model

was used in this experiment (Wan Nordini Hasnor, 2007; Rahim et al., 2008). Saliva-

coated glass beads of 3 mm diameter were used to mimic the hard tissue surfaces of the

tooth in the oral cavity. A modified Pasteur pipette served as a chamber where the glass

beads were then placed. The inflow and outflow of media from rubber tubings

connected to and from the chamber mimics the inflow and outflow of saliva in the oral

cavity. The flow rate of the media was controlled at 0.3 mL min-1

using a peristaltic

pump (Econo Pump, Bio Rad). Following the inoculation of 20 mL of 106 cell mL

-1 of

C. krusei suspension, the artificial mouth system was run overnight. The glass beads

were asceptically removed and immersed in separate appendorf vials containing 1 mL

of PBS. Each of the vials was placed in a sonicator for 60 seconds to dislodge the

48

adhered cells. The population of the adhered cells was determined by plating 100 µL of

the suspension obtained on YEPD agar plates. The total CFU/mL following a 24 hours

incubation period at 37°C was determined and recorded. This procedure was followed

closely to the steps outlined by Wan Nordini Hasnor (2007). Similar procedure was

repeated on the 1st to 4

th switched generations of cells.

3.2.9 Antifungal response of Candida krusei

3.2.9.1 Preparation of aqueous plant extracts (Fathilah, 2004)

3.2.9.1.1 Piper betle aqueous extract

Piper betle leaves were cleaned and the wet weight was taken. The leaves were

oven dried at 60 ºC for approximately 24 to 48 hours. The dried leaves were weighed

and recorded. 100 g of the leaves were cut into small pieces and put into a conical

flask. 2 L of distilled water were added and boiled until the volume was reduced to

half. Later, the decoction was filtered into a 500 mL beaker. The filtrate was re-boiled

until it was concentrated to a final volume of 100 mL. Finally, the concentrated extract

was freeze dried to produce Piper betle extract powder and kept in a dry cabinet in 30%

relative humidity (RH).

3.2.9.1.2 Nigella sativa aqueous extract

100 g of Nigella sativa seeds were cleaned and put in 2 L of distilled water. The

suspension of the mixture was boiled until the volume reduced to half. Next, filter

paper was used to separate debris. The filtrate was transferred into a 500 mL beaker.

The suspension was reboiled until it reached a final volume of 100 mL. The suspension

49

was freeze dried to produce Nigella sativa extract powder and kept in dry cabinet at

30% relative humidity (RH).

3.2.9.2 Susceptibility analysis

The susceptibility of C. krusei to CHX was determined following two methods;

the Kirby-Bauer disc diffusion test and broth dilution methods (Cappucino and

Sherman, 2005). According to the standard procedure of the Clinical and Laboratory

Standards Institute (CLSI), C. krusei suspension can be prepared by suspending 1 to 2

colonies of C. krusei grown on YEPD agar into 5 mL of 0.85% of sterile saline. The

optical density of the cell suspension was then standardised to an OD of 0.144 at 550

nm wavelength. 100 µL of the suspension was pipetted out and evenly swabbed on

Mueller-Hinton (MH) agar (BD, USA). Paper discs which have been impregnated with

120 µg (100 µL of 0.12%) CHX were carefully placed on to the swabbed MH agar

plate. The diameter of an inhibited growth zone surrounding the discs following an

overnight incubation at 37°C was then measured. On the same plate, discs incorporated

with 25 µg fluconazole and 1 µg voriconazole were used as the positive and negative

control, respectively. The susceptibility of Candida krusei to other antifungal agents

including amphotericin B (25 µg), nystatin (100 µg), Piper betle (1 mg) and Nigella

sativa (2 mg). The susceptibility of all switched cells towards these agents were also

determined and compared to the responses of the unswitched Candida krusei.

3.2.9.3 Determination of the minimal inhibitory concentration (MIC)

The MIC of CHX was determined using the broth microdilution method

(Cappucino and Sherman, 2005). A sterile 96-well microtiter plate was labelled W1 to

W7 horizontally and samples number vertically. Using a sterile pipette, 100 µL of

50

YEPD broth was added to W2 through W7 while 100 µL of CHX (0.12%) was added

into W1 and W2. The plate was slowly agitated to mix the content. Using a sterile

pipette, 100 µL of W2 was transferred to W3. Following thorough mixing, 100 µL of

W3 was transferred to W4 and the procedure was continued through W6. After mixing,

100 µL from W6 was discarded. W7 that received no CHX and W1 that has no

Candida krusei served as negative and positive control respectively for the experiment.

Lastly, 100 µL of Candida krusei suspension was added to W2 through W7 aseptically.

W7 that received no CHX served as a positive control. The plate was incubated

overnight at 37 ºC. The concentration of CHX in the well that showed no turbidity was

taken as the MIC of CHX towards Candida krusei. The MIC‟s of Candida krusei in the

unswitched and all switched generations to all other agents including amphotericin B,

nystatin, Piper betle and Nigella sativa were also determined.

3.2.9.4 Determination of the minimal fungicidal concentration (MFC)

The minimal fungicidal concentration (MFC) of CHX was determined following

the method described by Cappucino and Sherman (2005). MFC is referred as the

minimum fungicidal concentration at which 99% to 99.5% Candida krusei is killed.

This value was determined by inoculating 100 µL from the well of the previous

microtiter plate representing MIC on to YEPD agar plates respectively in triplicate. The

suspension was spread evenly on the agar surface and incubated for 18 to 24 hours at 37

ºC. Following incubation, the concentration from the plate which showed no growth of

Candida krusei was considered as the MFC concentration of CHX towards Candida

krusei. The MFC‟s of amphotericin B, nystatin, Piper betle and Nigella sativa were

determined following the same procedure.

51

3.2.9.5 Determination on the effect of CHX, Amphotericin B and Piper betle

aqueous extract on the growth curve of phenotype-switched Candida krusei

The effect of the respective antifungal agents on the growth curves of Candida

krusei was performed by monitoring the growth of the cells in a growth condition which

have been treated with the agents. Five sterilized Schott bottles were labeled with

unswitched (U), 1st switched generation (S1), 2

nd switched generation (S2), 3

rd switched

generation (S3) and 4th switched generation (S4) of Candida krusei respectively. 40 mL

of YEPD broth and 5mL of CHX stock (2 mg/mL) were added to each of the labeled

Schott bottle to give a final concentration of 0.2 mg/mL (sub-MIC of CHX). Following

this, 5 mL of each generation of Candida krusei (106 cell mL

-1) was then added

respectively into each of the Schott bottle according to the indicated labeled on the

bottle to give a total volume of 50 mL. The experiment was carried out in triplicates.

The suspension was vortexed vigorously for 30 seconds and the initial absorbance was

taken at OD550nm. The suspension was then incubated in a shaking water bath at 37 ºC.

The changes in OD of the growth suspension were recorded using spectrophotometer at

every one hour interval. A graph of increase in cell population against growth time was

plotted. The experiment was stopped when the stationary phase was achieved.

Similar protocol was repeated to determine the effect of the agents on the

growth process of the 1st to the 4

th switched cells with amphotericin B (250 µg/mL) and

Piper betle (60 mg/mL) by replacing the CHX (2 mg/mL). The final concentration of

amphotericin B and Piper betle aqueous extract at sub-MIC value of amphotericin B

(25 µg/mL) and Piper betle (6 mg/mL) were used in the experiment.

52

The growth rate constant (GR) and the generation time (GT) of Candida krusei

was finally calculated using the formula below:

GR = [(log10Nt2 – log10Nt1)2.303] / (t2-t1); t2>t1

GT = (log10Nt2 – log10Nt1) / log102

Nt1 = initial concentration

Nt2 = final concentration

t1 = initial time

t2 = final time

53

4.0 RESULTS

4.1 Biological characteristics of Candida krusei

4.1.1 Colony morphology

Sub-culturing of Candida krusei on YEPD agar was observed to produce colony

with undulate margins, circular forms and umbonate elevation. The surface appearance

of the unswitched Candida krusei was observed as dry and round surfaces with white to

cream colour. The nitrogen depleted growth environment induced by the addition of

Phloxine B had caused variations in the colony characteristics (Table 4.1, Figure 4.1).

The 1st and 2

nd switched generations were observed to have colonies with undulate

margin, circular form and umbonate elevation which were similar to the morphological

characteristics of the unswitched Candida krusei. The surface appearance of Candida

krusei in the 1st and 2

nd generations was observed as wrinkled appearance which was

absent in the unswitched Candida krusei. The 3rd

switched generation showed different

colony morphology with heavily wrinkled, myceliated surface appearance, lobate

margin, irregular form and umbonate elevation. The 4th

switched generation exhibited

similar surface appearance and elevation compared to the 3rd

switched generation

except for the filamentous margin and circular form.

The ability of Candida krusei to utilise chromogenic substrate and developing

colourised colony was determined by CHROMagar. This study has observed that all

colonies of unswitched and switched generations of Candida krusei grown on

CHROMagar exhibited pink colour colonies with pale border, dry and rough surface

appearances, undulate margin, circular form and umbonate elevation.

54

4.1.2 Recovery population of phenotypic switched Candida krusei

Comparative to the unswitched Candida krusei, the recovery population in terms

of CFU/mL showed that the 3rd

switched generation has the highest population recovery

of 85.7% followed by the 4th

generation at 70.8% and 46.6% for the 1st switched

generation. The 2nd

switched generation recovered the lowest population of only 36.4%

(Table 4.1).

55

Figure 4.1: Colony morphology of the unswitched and switched Candida krusei. (A)

Unswitched, (B) 1st switched generation, (C) 2

nd switched generation, (D) 3

rd switched

generation and (E) 4th

switched generation.

A

B C

D E

56

Table 4.1: The characteristics of growth colonies of the unswitched and all switched

generations of Candida krusei. Note: The terminologies used were according to

Samaranayake et al. (1994), Vargas et al. (2004) and Cappucino and Sherman (2005).

Growth

generation

Colony characteristics Percentage

of

Recovered

Population

(%)

Surface

Appearance

Margins Forms Elevation

Unswitched

Dry and

rough

Undulate Circular Umbonate 100.0

1st

switched Dry, rough

and wrinkled

Undulate Circular Umbonate 46.6

2nd

switched Dry, rough

and wrinkled

Undulate Circular Umbonate 36.4

3rd

switched Dry, rough,

heavily

wrinkled and

myceliated

Lobate Irregular Umbonate 85.7

4th

switched Dry, rough,

heavily

wrinkled and

myceliated

Filamentous Circular Umbonate 70.8

57

4.1.3 Biochemical validation of Candida krusei

API 20 C AUX identification system was used to determine on the ability of

Candida krusei to utilise substrates as a source of carbon. Results obtained indicated

that the unswitched and all switched generations of Candida krusei were able to ferment

only glucose (Figure 4.2). The BIOLOG identification system used in the study was

based on the principle of substrate-enzyme interactions. The unswitched and all

switched generations of Candida krusei were shown to be able to ferment N-acetyl-D-

glucosamine and α-D-glucose. Except for the unswitched and 1st generation which also

fermented γ-aminobutyric acid (GABA), the 2nd

, 3rd

and 4th

switched generations

responded negatively towards the fermentation of γ-aminobutyric acid.

Figure 4.2: Biochemical test using API 20 C AUX. Candida krusei was shown to

positively fermented glucose as indicated by the hazy lines in the well within the yellow

box.

4.1.4 Cell morphology of Candida krusei

Examination of prepared slides under 100 x magnification using light

microscope following simple staining showed that the cells of the unswitched and

switched Candida krusei have pseudohyphae (Figure 4.3). Blastoconidia were present

and observed as oval to elongated shape with the presence of verticillate branches.

58

Figure 4.3: Unswitched and switched Candida krusei examined at 100 x magnification

using a light microscope; (A) unswitched, (B) 1st switched generation, (C) 2

nd switched

generation, (D) 3rd

switched generation and (E) 4th switched generation.

4.1.5 Ultrastructural characteristics of Candida krusei

Scanning electron micrographs of the unswitched and all switched generations

of C. krusei were observed as branched cells with elongated pseudohyphae and

elongated to ovoidal blastoconidia and budding off in verticillate branch. The surface

appearance of the pseudohyphae was observed as smooth for the unswitched, 1st and 2

nd

B

C D

A

E

59

generations. However, the 3rd

and 4th

switched generations had exhibited changes on

the cell surface showing rough texture instead. In contrast to other switched

generations, the 4th

generation was observed to exhibit pimpled or punctate appearance

on the cell surface (Soll, 1992). The dimension of the 1st switched generation

pseudohyphae was found to be approximately 5.0-11.0 x 3.0-4.0 µm whereby the

pseudohyphae of the 2nd

switched generation was identified to be the most extended

compared to other generations with dimension of 5.0-15.0 x 2.0-4.0 µm. The size of

pseudohyphae of the 3rd

switched generation was determined as approximately 3.0-7.0 x

2.0-3.0 µm. The smallest pseudohypae was observed in the 4th

switched generation

with size ranging approximately 2.0-6.0 x 2.0-5.0 µm. In addition, the unswitched and

the 3rd

switched generations were observed to develop extracellular matrix which was

absent in the 1st, 2

nd and 4

th switched generations (Figure 4.4).

60

Figure 4.4: SEM micrographs of Candida krusei observed for the various growth

generations. (A) unswitched, (B) 1st switched generation, (C) 2

nd switched generation,

(D) 3rd

switched generation and (E) 4th switched generation (x 2000). Note: (A1)

Blastoconidia (A2) Pseudohyphae (A3) Extracellular matrix.

A3 A

B C

D E

A1

A2

61

4.1.6 Growth curves of Candida krusei

Figure 4.5 showed the growth curves plotted from the study. Descriptively, the

growth curves of the unswitched and all switched generations showed no significant

difference between all generations (p>0.05). However, slight deviations of growth

curves were observed among the generations. The early log phase of the unswitched

and all switched generations of Candida krusei were determined at three hours and the

middle of the log phase were achieved after seven hours of incubation.

The specific growth rate (GR) of Candida krusei was found to differ in

unswitched and all switched generations (Table 4.4). In the unswitched state, the GR of

Candida krusei was determined at 0.677 ± 0.021 h-1

. A slight decreased in GR was

observed in the 1st switched generation to 0.648 ± 0.131 h

-1 and determined as the

lowest GR among all generations of Candida krusei. The 2nd

switched generation had

showed an increased in the GR with 0.708 ± 0.021 h-1

which was also identified as the

highest GR. The GR was observed to decrease in the 3rd

(0.689 ± 0.132 h-1

) and 4th

switched generation (0.700 ± 0.100 h-1

).

Consequently, the generation time (GT) of all the respective curves also differ in

the unswitched and all switched generations (Table 4.4). In the unswitched state, the

GT was determined at 3.905 ± 0.031 h. A slight decreased in the GT was observed in

the 1st switched generation at 3.740 ± 0.101 h and determined as the lowest GT among

generations of Candida krusei. The 2nd

switched generation showed an increase in GT

to 4.085 ± 0.001 h which was also identified as the highest GT. The GT of the 3rd

switched generation was 3.976 ± 0.102 h whereby the 4th

switched generation was

4.041 ± 0.005 h.

62

Figure 4.5: The growth curve (GC) of Candida krusei. A comparison between

unswitched and all switched generations in untreated environment.

4.2 Adherence capacity of Candida krusei to saliva-coated glass surfaces

The ability of Candida krusei to adhere to the surfaces of saliva-coated glass

beads was recorded at (5.62 ± 2.95) x 102 CFU/mL. Figure 4.6 showed an increase to

(15.29 ± 10.32) x 102 CFU/mL in the 1

st switched generation compared to the

unswitched Candida krusei. A drastic increased in adherence capacity in the 2nd

switched generation to (154.0 ± 60.2) x 102 CFU/mL was observed. However, the

adherence capacity was reduced in the 3rd

and 4th switched generations of Candida

krusei to (18.76 ± 7.56) x 102 CFU/mL and (9.38 ± 0.37) x 10

2 CFU/mL, respectively.

4.5

5

5.5

6

6.5

7

7.5

0 2 4 6 8 10 12 14 16 18 20

Log

10C

FU

/mL

Time, hrs

Unswitched 1st switched 2nd switched 3rd switched 4th switched1st switched 2nd switched 3rd switched 4th switched

63

Figure 4.6: The adherence capacity of Candida krusei to saliva-coated glass surface.

The values were means ± standard deviation (n=9).

4.3 Antifungal responses of Candida krusei

4.3.1 Disinfectant

4.3.1.1 Susceptibility towards CHX

The degree of susceptibility towards CHX was found to differ in the unswitched

and all switched generations. In the unswitched state, Candida krusei was found to be

susceptible to CHX with a growth inhibition zone of 3.8 ± 0.1 cm and was the most

susceptible towards CHX compared to other generations. The degree of susceptibility

was gradually decreased in the 1st and the 2

nd switched generations with inhibition zone

of 3.5 ± 0.2 cm and 3.0 ± 0.1 cm, respectively (Figure 4.7). In the 3rd

and 4th

switched

generations, susceptibility towards CHX was observed to increase gradually with

inhibition zone of 3.4 ± 0.2 cm and 3.5 ± 0.1 cm, respectively. The MIC and MFC of

0 20 40 60 80 100 120 140 160 180

Unswitched

1st switched

2nd switched

3rd switched

4th switched

x 102 CFU/mL

4th

3rd

2nd

1st

64

Candida krusei at each switched generation were determined at 0.4 µg/µL (Table 4.2,

Table 4.3).

Figure 4.7: The susceptibility of Candida krusei in the unswitched and switched forms

towards CHX as determined using the disc diffusion method. The values were means ±

standard deviation (SD) (n=9).

4.3.2 Chemical-based agents

4.3.2.1 Susceptibility towards amphotericin B

The susceptibility of the unswitched Candida krusei towards amphotericin B

was recorded to have an inhibition zone diameter of 2.2 ± 0.1 cm. The degree of

susceptibility was gradually increased in the 1st, 2

nd and 3

rd switched generations with

inhibition zone of 2.3 ± 0.3 cm, 2.4 ± 0.1 cm and 2.6 ± 0.3 cm, respectively with the 3rd

switched generation was found to be the most susceptible towards amphotericin B

(Figure 4.8). The susceptibility was determined to decrease in the 4th switched

0 1 2 3 4 5

Unswitched

1st switched

2nd switched

3rd switched

4th switched

Inhibition zone (cm)

1st switched

2nd switched

3rd switched

4th switched

65

generations with inhibition zone of 2.4 ± 0.1 cm. The MIC and MFC of Candida krusei

at each switched generation were determined at 10 µg/mL (Table 4.2, Table 4.3).

Figure 4.8: The susceptibility of Candida krusei in the unswitched and switched forms

towards amphotericin B as determined using the disc diffusion method. The values

were means ± standard deviation (SD) (n=9).

4.3.2.2 Susceptibility towards nystatin

The susceptibility of unswitched Candida krusei towards nystatin was recorded

to have an inhibition zone of 2.4 ± 0.1 cm. The degree of susceptibility remained

unchanged in the 1st switched generation. However, the susceptibility was decreased in

the 2nd

switched generation with inhibition zone of 1.9 ± 0.2 cm respectively (Figure

4.9) and identified as the least susceptible towards nystatin. In the 3rd

and 4th

switched

generations, the susceptibility towards nystatin was observed to increase gradually with

inhibition zones of 2.3 ± 0.1 cm and 2.6 ± 0.1 cm respectively. The 4th

switched

generation was determined as the most susceptible among generations of Candida

0 0.5 1 1.5 2 2.5 3

Unswitched

1st switched

2nd switched

3rd switched

4th switched

Inhibition zone (cm)

4

th

3rd

2nd

1st

66

krusei. The MIC and MFC of Candida krusei at each switched generation were

determined at 50 unit/mL (Table 4.2, Table 4.3).

Figure 4.9: The susceptibility of Candida krusei in the unswitched and switched forms

towards nystatin as determined using the disc diffusion method. The values were means

± standard deviation (SD) (n=9).

4.3.3 Plant-based agents

4.3.3.1 Susceptibility towards Piper betle aqueous extract

The degree of susceptibility towards Piper betle aqueous extract was found to

differ in unswitched and all switched generations. In the unswitched state, Candida

krusei was found to be susceptible to Piper betle aqueous extract with inhibition zone

diameter of 2.2 ± 0.1 cm. Differently for the 1st switched generation where the degree

of susceptibility was found to increased with inhibition zone diameter of 2.3 ± 0.2 cm

(Figure 4.10). The susceptibility of Candida krusei towards Piper betle aqueous

extract was identified to decrease in the 2nd

switched generation with 2.1 ± 0.1 cm

0 0.5 1 1.5 2 2.5 3

Unswitched

1st switched

2nd switched

3rd switched

4th switched

Inhibition zone (cm)

1st

2nd

3rd

4th

67

inhibited zone. The susceptibility in the 3rd

switched generation remains unchanged

with inhibition zone of 2.1 ± 0.2 cm. The 4th

switched generation was found to have a

lowered susceptibility compared to the 2nd

and 3rd

generations with inhibition zone of

2.0 ± 0.2 cm which was the least susceptible towards Piper betle aqueous extract among

generations of Candida krusei. The MIC and MFC towards Piper betle aqueous extract

were determined at 12.5 mg/mL for unswitched and all switched generations of

Candida krusei (Table 4.2, Table 4.3).

Figure 4.10: The susceptibility of Candida krusei in unswitched and switched forms

towards Piper betle aqueous extract as determined using the disc diffusion method. The

values were means ± standard deviation (SD) (n=9).

4.3.3.2 Susceptibility towards Nigella sativa aqueous extract

From the analysis, all generations of Candida krusei were found to be resistant

to Nigella sativa aqueous extract (Table 4.2).

1 1.2 1.4 1.6 1.8 2 2.2 2.4

Unswitched

1st switched

2nd switched

3rd switched

4th switched

Inhibition zone (cm)

1st

2nd

3rd

d

4th

68

Table 4.2: The effect of phenotypic switching on the susceptibility of Candida krusei towards CHX, amphotericin B, nystatin, Piper

betle and Nigella sativa aqueous extract. (R) is referred to resistance. The inhibition zones were the mean ± standard deviation (SD)

with n=9. Note: Concentration of CHX is dependent to the concentration used in commercialized product. The concentration of

amphotericin B and Piper betle are dependent to the concentration used in the determination of susceptibility by CLSI whereby the

concentration of Piper betle is standardize to the concentration used in the determination of susceptibility of Candida krusei towards

Nigella sativa.

Type of

antimicrobial

agents

Active

ingredients Concentration

Growth generations

Unswitched 1st switched 2

nd switched 3

rd switched 4

th switched

Inhibition zone (cm)

Disinfectant CHX 1.2 µg/µL 3.8 ± 0.1 3.5 ± 0.2 3.0 ± 0.1 3.4 ± 0.2 3.5 ± 0.1

Chemical

based

Amphotericin B 100 µg/mL 2.2 ± 0.1 2.3 ± 0.3 2.4 ± 0.1 2.6 ± 0.3 2.4 ± 0.1

Nystatin 250 unit/mL 2.4 ± 0.1 2.4 ± 0.1 1.9 ± 0.2 2.3 ± 0.1 2.6 ± 0.1

Plant based

Piper betle 200 mg/mL 2.2 ± 0.1 2.3 ± 0.2 2.1 ± 0.1 2.1 ± 0.2 2.0 ± 0.2

Nigella sativa >200 mg/mL R R R R R

69

Table 4.3: The MIC and MFC of CHX, amphotericin B, nystatin, Piper betle and Nigella sativa aqueous extract towards the

unswitched and all switched generations of Candida krusei (n=9).

Growth

generations

CHX

(µg/µL)

Amphotericin B

(µg/mL)

Nystatin

(unit/mL)

Piper betle

(mg/mL)

Nigella sativa

(mg/mL)

MIC MFC MIC MFC MIC MFC MIC MFC MIC MFC

Unswitched 0.4 0.4 50 50 10 10 12.5 12.5 >200 >200

1st switched 0.4 0.4 50 50 10 10 12.5 12.5 >200 >200

2nd

switched 0.4 0.4 50 50 10 10 12.5 12.5 >200 >200

3rd

switched 0.4 0.4 50 50 10 10 12.5 12.5 >200 >200

4th

switched 0.4 0.4 50 50 10 10 12.5 12.5 >200 >200

70

4.4 Growth curves of unswitched and switched generations of Candida krusei

under treated environment

4.4.1 Disinfectant

4.4.1.1 Chlorhexidine (CHX)

Figure 4.11 showed the various growth curves plotted from the study. The

growth curves of the unswitched and all switched generations have showed no

significant difference among generations. However, slight deviations of growth curve

were observed among the generations. The early log phase of unswitched and all

switched generations of Candida krusei were determined at one hour incubation

whereby the middle log phase were observed at 5.5 hours of incubation.

The specific growth rate (GR) of CHX treated Candida krusei was found to

differ in unswitched and all switched generations. In the unswitched state, the GR of

Candida krusei was determined at 3.618 ± 0.051 h-1

. A gradual decreased in GR was

observed in the 1st and 2

nd switched generation with 0.597 ± 0.029 h

-1 and 0.339 ± 0.004

h-1

(43.2%) respectively. The 2nd

switched generation was determined as the lowest GR

among generations of Candida krusei. However, the degree of GR was found to

increase in the 3rd

switched generation with 0.592 ± 0.022 h-1

. A slight decreased in

GR was determined in the 4th switched generation with 0.566 ± 0.022 h

-1.

Consequently, the generation time (GT) of CHX treated Candida krusei was

also identified to differ in unswitched and all switched generations. In the unswitched

state, the GT of Candida krusei was determined at 3.566 ± 0.031 h. A slight decreased

in GT was observed in the 1st and 2

nd switched generation with 3.444 ± 0.035 h

and

71

1.953 ± 0.028 h respectively with the 2

nd switched generation was determined as the

lowest GT among generations of Candida krusei. The degree of GT was found to

increase in the 3rd

switched generation with 3.414 ± 0.022 h. However, the 4th

switched

generation was observed to encounter a slight decrease in GT with 3.267 ± 0.025 h.

Table 4.4 and figure 4.14 showed the GR and GT of unswitched and all

switched generations of Candida krusei were decreased. Among the generations, 2nd

switched generations were observed to be the most influence in CHX growth condition

with 52.1% reduction in GR. Whereby, the 1st switched generation was observed to be

the least influence by 7.9% reduction. These are similar to the GT where the most

influence generation was determine at the 2nd

switched generation whereas the least

influenced was the 1st switched generation with 52.2% and 7.9% GT reduction

respectively.

Figure 4.11: The growth curve (GC) of Candida krusei. A comparison between

unswitched and all switched generations in CHX treated environment.

4.5

5

5.5

6

6.5

7

7.5

0 2 4 6 8 10 12 14 16 18 20

Log

10C

FU

/mL

Time, hrs

Unswitched 1st switched 2nd switched 3rd switched 4th switched2nd switched1st switched 3rd switched 4th switched

4th switched

72

4.4.2 Chemical-based agent

4.4.2.1 Amphotericin B

Figure 4.12 showed the various growth curves plotted from the study. The

growth curves of the unswitched and all switched generations showed no significant

difference among all generations (p>0.05). Slight changes in the deviation of growth

curve were observed among the generations. The early log phase of unswitched and all

switched generations of Candida krusei were determined at one hour incubation

whereby the middle of the log phase were achieved after five hours incubation.

The growth rate (GR) of amphotericin B treated Candida krusei was found to

differ in unswitched and all switched generations. The GR of Candida krusei was

determined at 0.585 ± 0.013 h-1

in unswitched stage. An increased in GR was observed

in the 1st switched generation with 0.631 ± 0.014 h

-1 and determined as the highest

among generations. Following that, a decreasing in GR were observed in the 2nd

and 3rd

switched generations with GR of 0.585 ± 0.017 h-1

and 0.556 ± 0.021 h-1

respectively.

However, the 4th switched generation was observed to have an increased in GR with

0.606 ± 0.010 h-1

.

Consequently, the generation time (GT) of amphotericin B treated Candida

krusei was also identified to differ in unswitched and all switched generations. In the

unswitched state, the GT of Candida krusei was determined at 3.378 ± 0.051 h. An

increased in GT was observed in the 1st switched generation with 3.643 ± 0.042 h

and

determined as the highest GT among the generations. A decreased in GT was identified

in the 2nd

switched generation to 3.377 ± 0.054 h whereby the lowest GT was observed

73

in the 3rd

switched generation with 3.208 ± 0.073 h. However, the GT was found to

increase in the 4th switched generation with 3.498 ± 0.081 h.

Table 4.4 and figure 4.14 showed the GR and GT of unswitched and all

switched generations of Candida krusei were decreased. Among the generations, 3rd

switched generations were observed to be the most influenced in amphotericin B growth

condition with 19.3% reduction in GR. Whereby, the 1st switched generation was

observed to be the least influence by 2.6% reduction. These are similar to the GT where

the most influence generation was determine at the 3rd

switched generation whereas the

least influenced was the 1st switched generation with 19.3% and 2.6% GT reduction

respectively.

Figure 4.12: The growth curve (GC) of Candida krusei. A comparison between

unswitched and all switched generations in amphotericin B treated environment.

4.5

5

5.5

6

6.5

7

7.5

0 2 4 6 8 10 12 14 16 18 20

Log

10C

FU

/mL

Time, hrs

Unswitched 1st switched 2nd switched 3rd switched 4th switched2nd switched1st switched 3rd switched 4th switched

74

4.4.3 Plant-based extract

4.4.3.1 Piper betle aqueous extract

Figure 4.13 showed the various growth curves plotted from the study. The

growth curves of the unswitched and all switched generations has no significant

difference among all generations (p>0.05). However, slight deviations of growth curve

were observed among the generations. The early log phase of unswitched and all

switched generations of Candida krusei were determined at one hour incubation

whereby the middle of the log phase were achieved after seven hours incubation.

The growth rate (GR) of Piper betle treated Candida krusei was found to differ

in unswitched and all switched generations. In the unswitched state, the GR of Candida

krusei was determined at 0.560 ± 0.044 h-1

. A drastic decreased of GR was observed in

the 1st switched generation with 0.387 ± 0.053 h

-1 (30.9%) and was the lowest GR

among generations of Candida krusei (Figure 4.20). However, the 2nd

switched

generation has showed an increased in GR with 0.507 ± 0.031 h-1

(31%) followed by 3rd

and 4th

switched generations with GR 0.532 ± 0.032 h-1

(4.9%) and 0.586 ± 0.132 h-1

(10.2%) respectively. The GR of the 4th

switched generation was determined as the

highest among unswitched and switched generations of Candida krusei.

The generation time (GT) of Piper betle treated Candida krusei was also

identified to differ in unswitched and all switched generations. In the unswitched state,

the GT of Candida krusei was determined at 3.233 ± 0.321 h. The GT was observed to

decreased in the 1st switched generation with 2.235 ± 0.231 h and determined as the

lowest GT between generations. The 2nd

switched generation showed an increased in

75

GT with 2.923 ± 0.221 h followed by the 3

rd and 4

th switched generations with GR

3.069 ± 0.234 h and 3.382 ± 0.312 h respectively. The 4th

switched generation was

determined as the highest GT among unswitched and all switched generations of

Candida krusei.

Table 4.4 and figure 4.14 showed the GR and GT of unswitched and all

switched generations of Candida krusei were decreased. Among the generations, 1st

switched generation was observed to be the most influence in Piper betle growth

condition with 43.4% reduction in GR. Whereby, the 4th

switched generation was

observed to be the least influenced by 16.3% reduction. These are similar to the GT

where the most influenced generation was determine at the 3rd

switched generation

whereas the least influenced was the 1st switched generation with 40.2% and 16.3% GT

reduction respectively.

Figure 4.13: The growth curve (GC) of Candida krusei. A comparison between

unswitched and all switched generations in Piper betle aqueous extract treated

environment.

4.5

5

5.5

6

6.5

7

7.5

0 2 4 6 8 10 12 14 16 18 20

Log

10C

FU

/mL

Time, hrs

Unswitched 1st switched 2nd switched 3rd switched 4th switched2nd switched1st switched 3rd switched 4th switched

76

Unswitched

1st switched

2nd

switched

3rd

switched

4th

switched

Figure 4.14: The growth curve (GC) of unswitched and switched Candida krusei of

untreated ( ), CHX ( ), amphotericin B ( ) and Piper betle ( ) treated

growth environment.

5

5.5

6

6.5

7

7.5

0 2 4 6 8 10 12 14 16 18 20

log

10C

FU

/mL

Time, hrs

5

5.5

6

6.5

7

7.5

0 2 4 6 8 10 12 14 16 18 20

log

10C

FU

/mL

Time, hrs

5

5.5

6

6.5

7

7.5

0 2 4 6 8 10 12 14 16 18 20

log

10C

FU

/mL

Time, hrs

5

5.5

6

6.5

7

7.5

0 2 4 6 8 10 12 14 16 18 20

log

10C

FU

/mL

Time, hrs

5

5.5

6

6.5

7

7.5

0 2 4 6 8 10 12 14 16 18 20

log

10C

FU

/mL

Time, hrs

1st switched

2nd switched

3rd switched

4th switched

1st switched

2nd switched

77

Table 4.4: The changes in the generation times (GT) and specific growth rate (GR) of unswitched and all switched generations of

Candida krusei when their growth were perturbed with the introduction of CHX, amphotericin B and Piper betle. The concentrations

used in the study were dependent to the concentration of sub-MIC.

Growth conditions growth rate (GR)

and Generation times

(GT)

Growth generations

Unswitched 1st switched 2

nd switched 3

rd switched 4

th switched

Untreated

GR (h-1

) 0.677 ± 0.021 0.648 ± 0.131 0.708 ± 0.021 0.689 ± 0.132 0.700 ± 0.100

GT (h) 3.905 ± 0.031 3.740 ± 0.101 4.085 ± 0.001 3.976 ± 0.102 4.041 ± 0.005

CHX

GR (h-1

) 0.618 ± 0.051 0.597 ± 0.029 0.339 ± 0.004 0.592 ± 0.022 0.566 ± 0.022

GT (h) 3.566 ± 0.031 3.444 ± 0.035 1.953 ± 0.028 3.414 ± 0.022 3.267 ± 0.025

Amphotericin B

GR (h-1

) 0.585 ± 0.013 0.631 ± 0.014 0.585 ± 0.017 0.556 ± 0.021 0.606 ± 0.010

GT (h) 3.378 ± 0.051 3.643 ± 0.042 3.377 ± 0.054 3.208 ± 0.073 3.498 ± 0.081

Piper betle

GR (h-1

) 0.560 ± 0.044 0.387 ± 0.053 0.507 ± 0.031 0.532 ± 0.032 0.586 ± 0.132

GT (h) 3.233 ± 0.321 2.235 ± 0.231 2.923 ± 0.221 3.069 ± 0.234 3.382 ± 0.312

The values were the mean ± standard deviation (SD) of triplicates from three determinations (n=9).

78

5.0 DISCUSSION

In this study, phenotypic switching of Candida krusei was induced by the

addition of phloxine B (tetrabromotetrachlorofluorescein) which acts as switching

detecting agent for Candida sp. The addition of phloxine B in the media has created a

nitrogen suppressed growth condition for Candida krusei (Coote, 2001). The chemical

compounds in Phloxine B dye have been shown able to facilitate the detection of mutant

yeast that lack the capability synthesizing purine and pyramidine bases or amino acids.

It has also been reported that phloxine B is not a growth inhibitor, but it promotes death

of candidal yeast under nitrogen limitation conditions (Middelhoven et al., 1976). Thus,

the application of phloxine B in the growth environment had caused Candida krusei

cells to switch in order to overcome the killing factors provided by the dye.

Candida krusei in this study exhibited characteristic of cream to whitish colour,

dry and rough surface appearance with undulate margin, circular forms and umbonate

elevation when cultured on YEPD agar. Samaranayake and Samaranayake (1994) had

earlier described the colony morphology of Candida krusei as matt, rough surface

appearance with cream to whitish colour on SDA. Both observations did not conform

to the characteristics given in the ATCC manual which described Candida krusei as

butyrous surface with entire margin on SDA. Variations in the colony characteristics

may be due to the different growth medium used. This may affect the development of

colony morphology of Candida krusei. YEPD agar is a medium containing rich mineral

of zinc and able to suppress the expression of phenotype. Nevertheless, it does not

influence the switching system of Candida sp. (Odds et al., 1989) and therefore the

usage of YEPD in the study did not interfere with the phenotypic switching

determination.

79

The colony morphology in each generation of switched Candida krusei was

observed different in terms of surface appearance, margin, form and elevation. This

finding was similar to the reports on Candida albicans and Candida glabrata where

various colony morphology phenotypes were observed at different growth generations

including smooth, myceliated and wrinkled surface appearance (Vargas et al., 2004). In

our study, we had observed the transition in the colony morphology characteristics

between the 2nd

and 3rd

switched generations which are similar to the reported findings

on Candida albicans which showed to exhibit predominant transition between

unmyceliated to myceliated colony (Soll et al., 1987). Phenotypic switching

phenomenon could also occur after a prolonged incubation (Slutsky et al., 1985; Slutsky

et al., 1987) which may enhance the development of different colony morphology of

Candida krusei. These different switched phenotypes act as a survival strategy of

Candida krusei, as different phenotype serve a different role in providing adaptability

and survivability at different condition (Soll, 1992).

CHROMagar is a chromogenic medium which is widely used in the

identification and detection of yeasts including Candida krusei (Hospenthal et al.,

2006). The superiority to inhibit the growth of bacterial strains was determined to be

higher compared to SDA which is generally used in the identification of candidal

species (Sivakumar et al., 2009). From the study, the colonies of Candida krusei were

determined to grow pink in colour with pale border, dry and rough surface appearances,

undulate margin, circular form and umbonate elevation which are similar to the finding

by Hospenthal et al. (2002). The colourization of the colony is due to the reaction of

specific enzymes produced by Candida krusei towards chromogenic substrates yielding

microbial colonies expressing specific pigmentation, hence allowing the confirmation of

80

the species by the detection of colour and colony morphology of the candidal strains

(Sivakumar et al., 2009).

The recovery population determined the sustainability of each switched

generation of Candida krusei under suppressed growth environment. In our study, the

recovery population of the 3rd

switched generation was found to be the highest

recovered at 85.7% followed by the 4th

generation at 70.8%. 1st switched generation

was identified to have recovery percentage of 46.6% and the 2nd

switched generation

was determined to have the least recovery population with only 36.4%. The difference

on the recovery population was suggested to occur due to the different phenotype

plasticity among switched generations. Similar findings on the various population

recovery was reported where different switched generations possessed different

percentage recovery population, thus, suggested that different generations represent

different survival ability and stability due to the suppressed environment (Lackhe et al.,

2000).

From the study, the unswitched and all switched generations of Candida krusei

were identified to ferment only glucose out of 19 other substrates including glucose,

glycerol, 2-keto-D-gluconate, L-arabinosa, D-xylose, adonitol, xylitol, galactose,

inostol, sorbitol, α-methyl-D-glucoside, N-acetyle-D-glucosamine, cellobiose, lactose,

maltose, sucrose, trehalose, melezitose and raffinose. According to Melville and

Russells (1975), Candida krusei can ferment dextrose, producing acid and gas. This

phenomenon was reported similar with the finding by Samaranayake and Samaranayake

(1994) where Candida krusei was reported to ferment only glucose out of a large panel

of carbohydrates. The unswitched and all switched Candida krusei were observed

81

fermenting N-acetyl-D-glucosamine (C8H15NO6) as a carbon source. This may suggest

that all generations of Candida krusei are able to ferment N-acetyl-D-glucosamine

which is a derivative of the monosaccharide glucose. Candida krusei was also

determined as a pathogenic microorganism which is able to grow in vitamin-free media

(Odds, 1988). From the study, γ-aminobutyric acid (GABA) was found to be one of the

nutrient sources for the unswitched and 1st switched generations of Candida krusei.

According to Kumar and Punekar (1997), most yeasts and fungi utilise GABA as a

source of carbon and nitrogen. This substrate was identified as an important agent

which associate to the sporulation and spore metabolism of the yeast. Information on

the role of GABA in fungal biology is gradually increasing.

Based on light microscope observation, in general Candida krusei forms

elongated pseudohyphae with elongated to ovoidal blastoconidia and budding off

verticillate branch. These characteristics conform to the description on cellular

characteristics of Candida krusei by Samaranayake and Samaranayake (1994). Candida

krusei was also described in the ATCC manual as „long grain rice‟ shaped yeast with

branched pseudohyphae and elongated blastoconidia.

However, based on SEM micrograph, some variations in the cell morphology

characteristics were observed throughout different switched generations, which could

occurred due to some environmental constrains during the blastoconidia-hyphae

transitions. The transition of smooth to pimpled and punctate morphology in the 3rd

to

the 4th switched generation of Candida krusei observed in our study was similar to the

response as the transition of white to opaque cell in Candida albicans switched

generations. According to Soll (1992), the formation of pimpled and punctate

82

characteristic observed in the ultrastructure of candidal cells could be an outcome of

blastoconidia and pseudohyphae maturity in each level of the switched generations. In

addition, the variant colony morphologies have been described in several reports to be

dependent on the proportion and distribution of blastoconidia and pseudohyphae. Their

presence could have led to the changes in the colony morphology of the switched

Candida krusei (Vargas et al., 2004).

In the study, 2nd

switched generation of Candida krusei was identified to be

more extended compared to other generation. According to Anderson and Soll (1987),

this extension which also occurs among switched Candida albicans is due to the

distribution of actin granules which is mostly found on the apex of the pseudohyphae

and the generations of various characteristics of pseudohyphae were dependent on the

pattern of actin granule distribution between growing blastoconidia and pseudohyphae

in the candidal strains. It is also suggested that the hyphae-specific genes may be

transiently recruited among switched Candida krusei as an adaptation to the

environmental changes which then led to the different dimension and size of the cell of

Candida krusei. Thus, hyphae-specific function and hyphae specific gene expression

were identified to play an important role in generating unique phenotype at different

switched generation of Candida krusei.

From the susceptibility tests, all switched generations of Candida krusei were

found to be susceptible to CHX. According to several researches, CHX affects the

plasma membrane of the candidal cell by non-specific binding to the negatively charged

protein and phospholipid moieties of the cell wall. This binding will then alter the

cellular membrane structure and interfere with the cellular osmotic balance that lead to

83

the susceptibility of candidal strains (Freitas et al., 2003; Bonacorsi et al., 2004;

Veerman et al., 2004) towards CHX. In addition, this study demonstrated that the

unswitched and all switched generations of Candida krusei were susceptible to

amphotericin B. According to Anil (2002) and Williams et al. (2011), amphotericin B

is grouped as polyene which acts as a broad spectrum of fungicidal and fungistatic.

This polyene affects the composition of the sterol on the cell wall of the target cells

which then damage the cell walls. The damaging caused potassium ions and glucose to

be released out from the cell, disturbing the glycolysis which finally inhibits the growth

of the candidal cells.

The unswitched and all switched Candida krusei were found to be susceptible to

nystatin. This sensitivity occurred due to the mechanism of altering the cell

permeability of candidal strains that induce cell porosity (Kerridge, 1986). The

interaction between nystatin and ergosterol component within the cell membrane

influence the cell permeability due to the lost of cytoplasmic membrane which then lead

to the mortality of Candida krusei (Williams et al., 2011).

The study has shown that the unswitched and all switched Candida krusei were

also susceptible to Piper betle aqueous extract. Piper betle was classified as antifungal

agents having the potential of damaging the cell membrane of the candidal species

which lead to the lost of the cell viability and leakage of the intracellular constituents

(Indu and Ng, 2002; Guha, 2006). The active components such as hydroxichavicol,

stearic acids and hydroxyl fatty acids esters has extensively reported as the antibacterial

and antifungal agents and widely used in traditional therapeutic (Pauli et al., 2002;

Nalina and Rahim, 2007).

84

In this study, some variations in the degree of susceptibility between the various

switched generations displayed the responses of the switched cells to survive and attain

overall fitness. In other words, as described by Vargas (2004), when a cell undergo

switching, many of its features such as cell physiology, antigenicity of the cell surface,

the composition of its basic molecules like protein, lipid and sugar may be altered and

stimulated in the attempt to achieve the best adaptability to the environmental constrain.

All generations of Candida krusei in our study had shown the ability to adhere

to the surfaces of saliva-coated glass beads. This finding was reported by

Samaranayake et al. (1994) as Candida krusei was found to adhere higher on inert

surfaces compared to the buccal epithelial cells (BEC). In addition, Candida krusei was

also reported to exhibit high hydrophobicity ability which encouraged adherence. The

hydrophobicity of Candida krusei was identified to have 5-fold greater than Candida

albicans (Samaranayake et al., 1994). Nevertheless, our study had found that the

adherence ability varied among switched generations of Candida krusei. The adherence

of all switched Candida krusei were found to be higher compared to the unswitched

generation. The 2nd

switched generation was determined to have the highest adherence

ability followed by 3rd

, 1st and 4

th switched generation. According to Jones et al.

(1994), phenotype switched can change the ability of Candida sp. to attach to a surface.

In addition, the type or form of hyphae following phenotypic switch has been found to

influence the adherence of candidal cells to inert surfaces (Jones et al., 1994). This

might explain the highest adhering ability exhibited by the 2nd

switched generation cells

(Anderson and Soll, 1987).

85

From the study, the unswitched and all switched generations of Candida krusei

showed varying degrees of responses following exposure to antimicrobial agents. The

unswitched and all switched generations were observed to influence in antimicrobial

presenting growth environment. The reducing in GR and GT in all growth generations

indicated that the microbial agents showed efficacy in the treatment of Candida krusei.

These different responses in the growth activities were determined as an outcome of

phenotypic switching. Regulation of the growth activities could be an attempt to

maintain the fitness of the cells to survive under adverse conditions (Vargas et al.,

2004). According to Cowen et al. (2001), an observation on the ability of Candida

albicans to adapt to the inhibitory concentration in fluconazole treated environment

found that the strain was producing different genes expression that involve in drug

resistance which then lead to the variability in the generation time (GT) of some isolates

that had been generated from one progenitor which suggested similar response occurred

in the phenotypic switching of Candida krusei presented in this study.

86

6.0 CONCLUSION

This study has determined the phenotypic switching capability of Candida

krusei which contributed to the changes in the biological characteristics as well as the

adherence capacity towards hard surface. The various in responses among unswitched

and switched Candida krusei towards CHX, nystatin, amphotericin B and plant extracts

(Nigella sativa and Piper betle) indicated that the phenotypic switching affects the

susceptibility of the candidal strains. Thus, it is concluded that the phenotypic

switching of Candida krusei leads to the pathogenic property in the oral cavity.

87

7.0 FUTURE STUDIES

1) To determine the biological characteristics of phenotypic switched

genetically Candida krusei.

2) To determine specific genes which enhance the phenotypic switching

properties of Candida krusei.

3) To characterize mono-species biofilm (MSB) and dual-species biofilm

(DSB) of phenotypic switched Candida krusei and non-krusei on denture

acrylic surface.

4) To determine the factors affecting biofilm formation of MSB and DSB of

phenotypic switched Candida krusei and non-krusei.

5) To evaluate the consequences of MSB and DSB of phenotypic switching

Candida krusei and non-krusei on the susceptibility towards active

component of Piper betle aqueous extract.

88

APPENDIX 1

Proceeding. 30th symposium of Malaysian Society for Microbiology, Kuantan, 16

th to

19th August 2008.

89

90

91

92

APPENDIX 2

Abstract. The 2nd

Thailand International Conference on Oral Biology, TiCOB, Thailand,

2008.

93

APPENDIX 3

Abstract. IADR PAPF/APR, Wuhan, China, 22th to 24

th September 2009.

94

APPENDIX 4

Abstract. 3rd

Dental Postgraduate Students Seminar 2010, Kuala Lumpur, 29th to 30

th

June 2010.

95

APPENDIX 5

Abstract. My1Bio Conference 2010, Kuala Lumpur, 30th to 31

th October 2010.

96

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